Antioxidants, preparation methods and uses

ABSTRACT

The invention concerns compounds of general formula (I) (wherein R and R′ represent an alkyl radical or an aryl group and R″ is hydrogen or a CO—R 1  group wherein R 1  is an alkyl radical or an aryl group; and the dimers formed by the disulphide bond from one and/or the other of the two sulphur atoms of the compounds of general formula (I); and the corresponding thiazolidin forms), the methods for preparing them and uses thereof. More particularly, it concerns the use of said compounds as antioxidant agents, in particular for preparing medicines designed to increase the intracellular and/or extracellular level of glutathione (GSH).

The present invention relates to novel compounds possessing antioxidantactivity, to their processes of preparation and to their uses, inparticular in the preparation of medicaments intended to increase theintracellular and/or extracellular level of glutathione (GSH).

An increasing number of studies are showing that reactive oxygen speciesplay an important role in many biological processes and in particular inthe development of many human pathologies and more particularly inretroviral infections by the human immunodeficiency virus (HIV).

Reactive oxygen species (ROS, superoxide ion, hydrogen peroxide,hypochlorous ion, hydroxyl radicals, and the like) are natural productsof variable origin; they originate from activated inflammatory cells,cells which metabolize xenobiotics or cells exposed to specificenvironmental milieus, such as cigarette smoke.

Various substances are known for being active in scavenging thesereactive oxygen species at the intracellular or extracellular level.

Mention may be made, for example, of glutathione, which is a tripeptide(L-γ-glutamyl-L-cysteinylglycine, GSH) found in all eukaryote cells. Itis synthesized and decomposed in the cell, mainly via its reduced form,GSH. This reduced tripeptide, which participates in many cell functions,such as, for example, protein and nucleic acid synthesis, and thetransportation of amino acids, constitutes the main mechanism forintracellular defense against oxidative stress. The factors whichpromote the formation of reactive oxygen species lead to the consumptionof the glutathione reserves.

N-Acetyl-L-cysteine (NAC) has been known for many years as a medicamentfor the cornea, as an antidote to poisoning by acetaminophen and as amucolytic agent by cleaving the disulfide bonds in mucoproteins. BecauseNAC is used therapeutically in many pathologies in which oxidizingagents seem to play a role, it has been suggested that it operates as anantioxidant. The mechanism of action of NAC is based on its ability toreduce extracellular cystine to cysteine or to be a source of SH (thiolfunctional group) metabolites. As a source of SH groups, NAC stimulatesthe synthesis of GSH, enhances the glutathione S-transferase activity,promotes detoxification and acts directly on reactive oxidizing species.NAC and, by extension, the acylated variants of the amino acidL-cysteine are an excellent source of SH groups and are converted in thebody into metabolites capable of stimulating the synthesis ofglutathione, thus promoting detoxification and acting directly asscavengers of reactive oxygen species.

In view of the central role of GSH in cell detoxification mechanisms,numerous alternatives targeted at raising its intracellular level havebeen envisaged as adjuvant therapeutic strategy in numerous humanpathologies and more particularly in infection by the humanimmunodeficiency virus (HIV) in recent years (WO 92/21368; WO 95/10268;U.S. Pat. No. 4,927,808; 5,580,577). The problem was first of allapproached by seeking to supplement the deficient molecule. Subsequentapproaches were targeted at feeding the γ-glutamyl cycle. Thus,L-cysteine, GSH itself, NAC, 2-oxothiazolidine-4(R)-carboxylic acid(OTC) and cysteamine (MEA), the formulae of which are reproduced below,were tested as potential adjuvants for antiretrovirals in the treatmentof infections by the HIV.

The beneficial effects of these molecules have been limited in vivo bylow bioavailability, excessively rapid metabolization and insufficientconcentrations which can be administered. NAC, for example, is arelatively labile molecule which, during its rapid decomposition,releases compounds comprising malodorous sulfides, such as H₂S. Thisproblem of instability has limited the use of NAC or other compoundsproviding a source of —SH, such as L-cysteine or its acylated variants,in the preparation of formulations which can be used in pharmacology,dermatology or cosmetics.

The inventors have now developed novel, compounds, capable of acting onthe intracellular or extracellular level of glutathione, which do notexhibit the instability which was found for the known products. This iswhy a subject matter of the present invention is the compounds offollowing general formula (I):

and in which:

-   -   R and R′ independently represent a linear or branched C₁-C₇        alkyl radical or an aryl group which is unsubstituted or        substituted by one or more radicals chosen from halogens, linear        or branched C₁-C₃ alkyl radicals and —OH (hydroxyl) radicals;    -   R″ is hydrogen or a CO—R¹ group in which R¹ is a linear or        branched C₁-C₇ alkyl radical or an aryl group which is        unsubstituted or substituted by one or more radicals chosen from        halogens, linear or branched C₁-C₃ alkyl radicals and —OH        radicals;        and the dimers formed by a disulfide bridge from one and/or        other of the two sulfur atoms of the molecule of general formula        I composed of the R″ radicals or of the R′ CO— radicals of the        two molecules, and the corresponding thiazolidine forms.

The R, R′ and R″ alkyl radicals are preferably C₁-C₃ radicals. Thehalogens are preferably chlorine and fluorine.

In these compounds, NAC and MEA are combined and are or are notprotected by biolabile groups.

According to a preferred embodiment, the present invention relates to acompound of general formula I such that R is a methyl group (—CH₃). In amore preferred embodiment, the present invention relates to a compoundof general formula I such that R and R′ are methyl groups (—CH₃). In thepreferred embodiment, the invention relates to the compound known asN-(N-acetyl-L-cysteinyl)-S-acetylcysteamine, known below as I-152, suchthat, in the general formula I, R and R′ are methyl groups (—CH₃) and R″is hydrogen.

This compound is particularly advantageous for its properties, whichrender it active in the treatment and/or prevention of pathologies ordisorders related to an intra- and/or extracellular depletion inglutathione, in particular the treatment of viral infections and moreparticularly infections by the human immunodeficiency virus (HIV).

According to another preferred embodiment, the invention relates to thecompound known as N-(N,S-bisacetyl-L-cysteinyl)-S-acetylcysteamine,known below as I-176, such that, in the general formula I, R and R′ aremethyl groups (—CH₃) and R″ is an acetyl group (—COCH₃).

According to another preferred embodiment, the invention relates to thecompound known asN-(N-acetyl-S-isobutyryl-L-cysteinyl)-S-acetylcysteamine, known below asI-177, such that, in the general formula I, R and R′ are methyl groups(—CH₃) and R″ is an isobutyryl group(—COCH (CH₃)₂).

According to another preferred embodiment, the invention relates to thecompound known asN-(N-acetyl-S-pivaloyl-L-cysteinyl)-S-acetylcysteamine, known below asI-178, such that, in the general formula I, R and R′ are methyl groups(—CH₃) and R″ is a pivaloyl group (—COC(CH₃)₃).

It is also one of the objects of the present invention to provide acompound of general formula I in which R is a methyl group (—CH₃) and R′is selected from the isopropyl group (—CH (CH₃)₂), the tert-butyl group(—C(CH₃)₃) and the phenyl group (—C₆H₅); such a compound preferablyexhibits an R″ group selected from hydrogen (—H); the acetyl group(—COCH₃), the isobutyryl group (—COCH(CH₃)₂), the pivaloyl group(—COC(CH₃)₃) or the benzoyl group (—CO—C₆H₅). More particularly, theinvention is targeted at providing the compound below known as:

-   -   I-188, such that, in the general formula I, R is a methyl group        (—CH₃), R′ is an isopropyl group (—CH(CH₃)₂) and R″ is hydrogen        (—H).    -   I-189, such that, in the general formula I, R is a methyl group        (—CH₃), R′ is an isopropyl group (—CH(CH₃)₂) and R″ is the        acetyl group (—COCH₃).    -   I-190, such that, in the general formula I, R is a methyl group        (—CH₃), R′ is an isopropyl group (—CH(CH₃)₂) and R″ is the        isobutyryl group (—COCH(CH₃)₂).    -   I-191, such that, in the general formula I, R is a methyl group        (—CH₃), R′ is an isopropyl group (—CH(CH₃)₂) and R″ is the        pivaloyl group (—COC(CH₃)₃).    -   I-192, such that, in the general formula I, R is a methyl group        (—CH₃), R′ is an isopropyl group (—CH(CH₃)₂) and R″ is the        benzoyl group (—CO—C₆H₅).    -   I-193, such that, in the general formula I, R is a methyl group        (—CH₃), R′ is a tert-butyl group (—C(CH₃)₃) and R″ is hydrogen        (—H)    -   I-194, such that, in the general formula I, R is a methyl group        (—CH₃), R′ is a tert-butyl group (—C(CH₃)₃) and R″ is the acetyl        group (—COCH₃).    -   I-195, such that, in the general formula I, R is a methyl group        (—CH₃), R′ is a tert-butyl group (—C(CH₃)₃) and R″ is the        isobutyryl group (—COCH(CH₃)₂).    -   I-196, such that, in the general formula I, R is a methyl group        (—CH₃), R′ is a tert-butyl group (—C(CH₃)₃) and R″ is the        pivaloyl group (—COC(CH₃)₃).    -   I-197, such that, in the general formula I, R is a methyl group        (—CH₃), R′ is a tert-butyl group (—C(CH₃)₃) and R″ is the        benzoyl group (—CO—C₆H₅).    -   I-198, such that, in the general formula I, R is a methyl group        (—CH₃), R′ is phenyl group (—C₆H₅) and R″ is hydrogen (—H).    -   I-199, such that, in the general formula I, R is a methyl group        (—CH₃), R′ is a phenyl group (—C₆H₅) and R″ is the acetyl group        (—COCH₃).    -   I-200, such that, in the general formula I, R is a methyl group        (—CH₃), R′ is a phenyl group (—C₆H₅) and R″ is the isobutyryl        group (—COCH(CH₃)₂).    -   I-201, such that, in the general formula I, R is a methyl group        (—CH₃), R′ is a phenyl group (—C₆H₅) and R″ is the pivaloyl        group (—COC(CH₃)₃).    -   I-202, such that, in the general formula I, R is a methyl group        (—CH₃), R′ is a phenyl group (—C₆H₅) and R″ is the benzoyl group        (—CO—C₆H₅).

It is also one of the objects of the present invention to provide acompound of general formula I which constitutes an intermediate in thesynthesis of the compound I-152, of its acylated derivatives or of itsanalogues. Thus, the invention also relates to the compound of generalformula I known as 10 in which R is a methyl group (—CH₃), R′ is theisopropyl group (—CH(CH₃)₂) and R″ is a trityl group. The invention alsorelates to the compound of general formula I known as 11 in which R is amethyl group (—CH₃), R′ is the tert-butyl group (—C(CH₃)₃) and R″ is atrityl group. The invention also relates to the compound of generalformula I known as 12 in which R is a methyl group (—CH₃), R′ is thephenyl group (—C₆H₅) and R″ is a trityl group.

It is also one of the objects of the present invention to provide acompound of general formula I in which R is an isopropyl group(—CH(CH₃)₂); such a compound of the invention is preferablycharacterized in that R′ is selected from the methyl group (—CH₃), theisopropyl group (—CH(CH₃)₂), the tert-butyl group (—C(CH₃)₃) and thephenyl group (—C₆H₅); such a compound preferably exhibits an R″ groupselected from hydrogen (—H), the acetyl group (—COCH₃), the isobutyrylgroup (—COCH(CH₃)₂), the pivaloyl group (—COC(CH₃)₃) or the benzoylgroup (—CO—C₆H₅). The invention is more particularly targeted atproviding the compound below known as:

-   -   I-203, such that, in the general formula I, R is an isopropyl        group (—CH(CH₃)₂), R′ is a methyl group (—CH₃) and R″ is        hydrogen (—H).    -   I-204, such that, in the general formula I, R is an isopropyl        group (—CH(CH₃)₂), R′ is a methyl group (—CH₃) and R″ is the        acetyl group (—COCH₃).    -   I-205, such that, in the general formula I, R is an isopropyl        group (—CH(CH₃)₂), R′ is a methyl group (—CH₃) and R″ is the        isobutyryl group (—COCH(CH₃)₂).    -   I-206, such that, in the general formula I, R is an isopropyl        group (—CH(CH₃)₂), R′ is a methyl group (—CH₃) and R″ is-the        pivaloyl group (—COC(CH₃)₃).    -   I-207, such that, in the general formula I, R is an isopropyl        group (—CH(CH₃)₂), R′ is a methyl group (—CH₃) and R″ is the        benzoyl group (—CO—C₆H₅).    -   I-208, such that, in the general formula I, R is an isopropyl        group (—CH(CH₃)₂), R′ is an isopropyl group (—CH(CH₃)₂) and R″        is hydrogen (—H).    -   I-209, such that, in the general formula I, R is an isopropyl        group (—CH(CH₃)₂), R′ is an isopropyl group (—CH(CH₃)₂) and R″        is the acetyl group (—COCH₃).    -   I-210, such that, in the general formula I, R is an isopropyl        group (—CH(CH₃)₂), R′ is an isopropyl group (—CH(CH₃)₂) and R″        is the isobutyryl group (—COCH(CH₃)₂).    -   I-211, such that, in the general formula I, R is an isopropyl        group (—CH(CH₃)₂), R′ is an isopropyl group (—CH(CH₃)₂) and R″        is the benzoyl group (—CO—C₆H₅).    -   I-214, such that, in the general formula I, R is an isopropyl        group (—CH(CH₃)₂), R′ is a tert-butyl group (—C(CH₃)₃) and R″ is        hydrogen (—H).    -   I-215, such that, in the general formula I, R is an isopropyl        group (—CH(CH₃)₂), R′ is a tert-butyl group (—C(CH₃)₃) and R″ is        the acetyl group (—COCH₃).    -   I-216, such that, in the general formula I, R is an isopropyl        group (—CH(CH₃)₂), R′ is a tert-butyl group (—C(CH₃)₃) and R″ is        the isobutyryl group (—COCH(CH₃)₂).    -   I-217, such that, in the general formula I, R is an isopropyl        group (—CH(CH₃)₂), R is a tert-butyl group (—C(CH₃)₃) and R″ is        the pivaloyl group (—COC(CH₃)₃).    -   I-218, such that, in the general formula I, R is an isopropyl        group (—CH(CH₃)₂), R′ is a tert-butyl group (—C(CH₃)₃) and R″ is        the benzoyl group (—CO—C₆H₅).    -   I-219, such that, in the general formula I, R is an isopropyl        group (—CH(CH₃)₂), R′ is a phenyl group (—C₆H₅) and R″ is        hydrogen (—H).    -   I-220, such that, in the general formula I, R is an isopropyl        group (—CH(CH₃)₂), R′ is a phenyl group (—C₆H₅) and R″ is the        acetyl group (—COCH₃).    -   I-221, such that, in the general formula I, R is an isopropyl        group (—CH(CH₃)₂), R′ is a phenyl group (—C₆H₅) and R″ is the        isobutyryl group (—COCH(CH₃)₂).    -   I-222, such that, in the general formula I, R is an isopropyl        group (—CH(CH₃)₂), R′ is a phenyl group (—C₆H₅) and R is the        pivaloyl group (—COC(CH₃)₃).    -   I-223, such that, in the general formula I, R is an isopropyl        group (—CH(CH₃)₂), R′ is a phenyl group (—C₆H₅) and R″ is the        benzoyl group (—CO—C₆H₅).

It is also one of the objects of the present invention to provide acompound of general formula I which constitutes an intermediate in thesynthesis of the compound I-152, of its acylated derivatives or of itsanalogues. Thus, the invention also relates to the compound of generalformula I known as 14 in which R is an isopropyl group (—CH(CH₃)₂), R′is a methyl group (—CH₃) and R″ is a trityl group. The invention alsorelates to the compound of general formula I known as 15 in which R isan isopropyl group (—CH(CH₃)₂), R′ is the isopropyl group (—CH(CH₃)₂)and R″ is a trityl group. The invention also relates to the compound ofgeneral formula I known as 16 in which R is an isopropyl group(—CH(CH₃)₂), R′ is the tert-butyl group (—C(CH₃)₃) and R′ is a tritylgroup. The invention also relates to the compound of general formula Iknown as 17 in which R is an isopropyl group (—CH(CH₃)₂), R′ is thephenyl group (—C₆H₅) and R′ is a trityl group.

The invention relates to the various compounds of the inventionmentioned above in the thiazolidine form. The invention relates moreparticularly to the compounds I-212 and I-213, the chemical formula ofwhich is given in scheme 5 below.

The invention relates to the compounds of formula I in the free form.

Another subject matter of the present invention is a process for thepreparation of the compounds of general formula (I) according toprocesses analogous to those used in peptide synthesis.

According to a first way of preparing the compounds of the invention,the process according to the invention comprises the following stages:

-   -   a) protection of the N-acyl-L-cysteine to provide the        N-acyl-S-trityl-L-cysteine compound;    -   b) coupling of the N-acyl-S-trityl-L-cysteine with the        S-acylcysteamine hydrochloride to provide the        N-(N-acyl-S-trityl-L-cysteinyl)-S-acylcysteamine compound.

Still according to a first way of preparing, the compounds of theinvention in thiazolidine form can be prepared according to the processwhich comprises the following stages:

-   -   a) protection of the N-acyl-L-cysteine to provide the        N-acyl-S-trityl-L-cysteine compound; then    -   b) coupling of the protected N-acyl-S-trityl-L-cysteine with        thiazolidine;

The compounds thus obtained can be subjected to the following stages of:

-   -   c) deprotection of said compound obtained in the preceding stage        b), then    -   d) releasing the free thiol of formula (I).

Thus, according to a preferred embodiment, the process for thepreparation of compounds of the invention comprises the stages of:

-   -   a) protection of the N-acyl-L-cysteine to provide the        N-acyl-S-trityl-L-cysteine compound;    -   b) coupling of the N-acyl-S-trityl-L-cysteine with the        S-acylcysteamine hydrochloride to provide the        N-(N-acyl-S-trityl-L-cysteinyl)-S-acylcysteamine compound;    -   c) S-detritylation reaction of the        N-(N-acyl-S-trityl-L-cysteinyl)-S-acylcysteamine compound, in        solution of methanol and chloroform, with a mixture of silver        nitrate, pyridine and methanol to provide the corresponding        silver sulfide;    -   d) suspending said corresponding silver sulfide in chloroform        and then releasing of the free thiol in the presence of HCl or        H₂S.

More particularly, the preparation of the compound I-152 of theinvention can be carried out by the preceding process; to do this, saidN-acyl-S-trityl-L-cysteine compound of stage a) isN-acetyl-S-trityl-L-cysteine and said S-acylcysteamine hydrochloride ofstage b) is S-acetylcysteamine hydrochloride.

An additional S-acylation stage can be attached to the preceding processfor the preparation of compounds according to the invention.

According to a second embodiment, the process according to theinvention, suitable for the preparation of the compound of generalformula I in which R=R′ and R″ is a hydrogen, comprises the followingstages:

-   -   a) esterification of the carboxyl functional group of        N-Boc-L-serine (1) with N-hydroxysuccinimide in        N,N-dimethylformamide (DMF) in the presence of        1,3-dicyclohexylcarbodiimide (DCC) to form the active ester        (1′); then,    -   b) in situ condensation of the active ester formed (1′) with        ethanolamine (2) to provide the compound        N-(N-Boc-L-seryl)-2-aminoethanol (3); then,    -   c) Mitsunobu reaction on the compound (3) with        triphenylphosphine and diisopropyl azodicarboxylate in the        presence of thiocarboxylic acid in tetrahydrofuran to provide        the compound N-(N-Boc-S-acyl-L-cysteinyl)-S-acylcysteamine (4);        in the context of the preparation of the compound I-512, the        thiocarboxylic acid is thioacetic acid; then,    -   d) deprotection of the compound (4) with trifluoroacetic acid.

In a specific embodiment, the invention relates to two processes for thepreparation of the compound I-152.

The first process for the preparation of the compound I-152 (scheme 1)corresponds to the process for the preparation of the compound ofgeneral formula (I) described above and involves correctly protectedL-cysteine. This preparation process is characterized in that itcomprises the following stages (i) of coupling ofN-acetyl-S-trityl-L-cysteine (7) with S-acetylcysteamine hydrochlorideto provide the compoundN-(N-acetyl-S-trityl-L-cysteinyl)-S-acetylcysteamine (8); then (ii) ofS-detritylation reaction of the compoundN-(N-acetyl-S-trityl-L-cysteinyl)-S-acetylcysteamine, in solution inmethanol and chloroform, with a mixture of silver nitrate, pyridine andmethanol to provide the corresponding silver sulfide (9); then (iii) ofsuspending said corresponding silver sulfide in chloroform; then (iv) ofrelease of the free thiol in the presence of HCl or H₂S.

-   -   Method A is based on the formation, in situ, of a mixed        anhydride by reaction of 7 with isobutyl chloroformate in AcOEt        in the presence of N-methylmorpholine (NMM). The anhydride is        subsequently condensed with S-acetylcysteamine, released from        its hydrochloride by NMM, to provide a8 with a yield, after        treatments, of 55%.    -   Method B uses, in situ, the N-succinimidyl active ester of 7        which, after condensation with S-acetylcysteamine, released from        its hydrochloride by NMM, makes it possible to obtain 8 with a        yield, after treatments, of 70%. The active ester was formed by        reaction of the preceding mixed anhydride (method A) with        N-hydroxysuccinimide in AcOEt.

The compound 8, in solution in methanol and chloroform, is subsequentlyS-detritylated by treatment with a mixture composed of silver nitrate,pyridine and methanol to provide the corresponding silver sulfide 9.This sulfide, which can be isolated, is then suspended in CHCl₃ and thenHCl (the use of H₂S leads to the same result) is added to release thefree thiol I-152.

The second process for the preparation of the compound I-152 (scheme 2)uses L-serine, N-protected by a t-butoxycarbonyl (Boc) (1), as startingmaterial.

This process is characterized in that it comprises the following stages(i) of activation of the carboxyl functional group of N′-Boc-L-serine(1) by N-hydroxysuccinimide in DMF in the presence of DCC; then (ii) ofin situ condensation of the active ester formed (1′) with ethanolamine(2) to provide the-compound N-(N-Boc-L-seryl)-2-aminoethanol (3); then(iii) of treatment according to a Mitsunobu reaction, modified by R. P.Volante (Tetrahedron Lett., 1981, 22, 3119-3122), ofN-(N-Boc-L-seryl)-2-aminoethanol with triphenylphosphine and diisopropylazodicarboxylate in the presence of thioacetic acid in tetrahydrofuran(THF) to provide the compoundN-(N-Boc-S-acetyl-L-cysteinyl)-S-acetylcysteamine (4). Thus, the fact ofconverting the alcohol of the L-serine into thioester, while retainingthe configuration of the asymmetric carbon, made possible conversion tothe L-cysteine series; then (iv) of deprotection ofN-(N-Boc-S-acetyl-L-cysteinyl)-S-acetylcysteamine with TFA; conventionaldeprotection of the N-Boc of 4 with TFA does not make it possible toisolate the corresponding amine formed 5, which is unstable under ouroperating conditions, but it makes it possible to synthesize thecompound I-152 by an intramolecular S→N transfer reaction of the acetylgroup of 5 via the corresponding thiazoline 6 (scheme 3). Suchtransfers, in particular on S-acetylcysteamine, have already beenobserved and studied (R. E. Barnett et al., and the references cited, J.Amer. Chem. Soc., 1969, 91, 2358-2369). These authors show that themechanism of transfer involves, under some pH conditions, the formationof an intermediate thiazoline which is subsequently hydrolyzed togenerate the N-acetylcysteamine. We find that the formation of I-152involves the same mechanism since we have isolated and identified thecyclic intermediate 6 which results from the N-deprotection of 4 via 5(scheme 3).

The S→N transfer reaction of the S-acyl on the cysteine residue makes itpossible to obtain the compound I-152 under the reaction conditionsindicated.

The present invention also relates to a process for the preparation ofthe compound of general formula I in which R and R′ are methyl groups(—CH₃) and R″ acyl groups; the preparation is carried out by S-acylationof the compound I-152 in solution in pyridine in the presence of ananhydride R₂O or of an acid chloride R—Cl, characterized in that R ischosen from the CO—R¹ group in which R¹ is a linear or branched C₁-C₇alkyl radical or an aryl group which is unsubstituted or substituted byone or more halogen atoms. Thus, the preparation of the compound I-176is carried out by S-acylation of the compound I-152, in solution inpyridine, with acetic anhydride. The preparation of the compound I-177is carried out by S-acylation of the compound I-152, in solution inpyridine, with isobutyryl chloride. The preparation of the compoundI-178 is carried out by S-acylation of the compound I-152, in solutionin pyridine, with pivaloyl chloride.

More generally, it is an object of the present invention to provide aprocess for the preparation of acylated analogues of the compound I-152or of its derivatives (scheme 4) by using method B of the first way ofpreparing the compound according to the invention described in scheme 1,route 1. This process comprises the stages of:

-   -   a) protection of N-acetyl-L-cysteine or N-isobutyryl-L-cysteine        to provide N-acetyl-S-trityl-L-cysteine (7) and        N-isbbutyryl-S-trityl-L-cysteine (13) respectively;    -   b) various couplings of (7) or (13) with S-acylcysteamine        hydrochlorides to provide various corresponding pseudopeptides.        Mention should be made, among these, of the compounds 10, 11 and        12, obtained from 7, and the compounds 14, 15, 16 and 17,        obtained from 13.

Alternatively, this process for the preparation of analogues of thecompound I-152 can be continued by a stage:

-   -   c) of S-detritylation reaction, as described above during the        preparation of I-152.

Thus, the S-detritylation reactions of the compounds 10, 11 and 12 arecarried out to give the corresponding thiol compounds I-188, I-193 andI-198. These compounds can be subjected to a stage:

-   -   d) of S-acylation to provide the compounds described above        I-189, I-190, I-191, I-192, I-194, I-195, I-196, I-197, I-199,        I-200, I-201, I-202.

Thus, the S-acylation reaction of I-188 makes it possible to obtain thecompounds I-189, I-190, I-191 and I-192. More particularly, theS-acetylation reaction of I-188 provides I-189, the S-isobutyrylationreaction of I-188 provides I-190, the S-pivaloylation reaction of I-188provides I-191 and the S-benzoylation reaction of I-188 provides I-192.

Thus, the S-acylation reaction of I-193 makes it possible to obtain thecompounds I-194, I-195, I-196 and I-197. More particularly, theS-acetylation reaction of I-193 provides I-194, the S-isobutyrylationreaction of I-193 provides I-195, the S-pivaloylation reaction of I-193provides I-196 and the S-benzoylation reaction of I-193 provides I-197.

Thus, the S-acylation reaction of I-198 makes it possible to obtain thecompounds I-199, I-200, I-201 and I-202. More particularly, theS-acetylation reaction of I-198 provides I-199, the S-isobutyrylationreaction of I-198 provides I-200, the S-pialoyation reaction of I-198provides I-201 and the S-benzoylation reaction of I-198 provides I-202.

Thus, the S-detritylation reactions of the compounds 14, 15, 16 and 17are carried out to give the corresponding thiol compounds I-203, I-208,I-214 and I-219. These compounds can be subjected to a stage:

-   -   d) of S-acylation to provide the compounds described above        I-204, I-205, I-206, I-207, I-209, I-210, I-211, I-215, I-216,        I-217 and I-218.

Thus, the S-acylation reaction of I-203 makes it possible to obtain thecompounds I-204, I-205, I-206 and I-207. More particularly, theS-acetylation reaction of I-203 provides I-204, the S-isobutyrylationreaction of I-203 provides I-205, the S-pivaloylation reaction of I-203provides I-206 and the S-benzoylation reaction of I-203 provides I-207.

Thus, the S-acylation reaction of I-208 makes it possible to obtain thecompounds I-209, I-210 and I-211. More particularly, the S-acetylationreaction of I-208 provides I-209, the S-isobutyrylation reaction ofI-208 provides I-210 and the S-benzoylation reaction of I-208 providesI-211.

Thus, the S-acylation reaction of I-214 makes it possible to obtain thecompounds I-215, I-216, I-217 and I-218. More particularly, theS-acetylation reaction of I-214 provides I-215, the S-isobutyrylationreaction of I-214 provides I-216, the S-pivaloylation reaction of I-214provides I-217 and the S-benzoylation reaction of I-214 provides I-218.

Thus, the S-acylation reaction of I-219 makes it possible to obtain thecompounds I-220, I-221, I-222 and I-223. More particularly, theS-acetylation reaction of I-219 provides I-220, the S-isobutyrylationreaction of I-219 provides I-221, the S-pivaloylation reaction of I-219provides I-222 and the S-benzoylation reaction of I-219 provides I-223.

As regards the compounds of the invention in the thiazolidine form, thelatter are preferably obtained by coupling N-acyl-S-trityl-L-cysteinewith thiazolidine according to method A described in scheme 1, route 1.The invention relates more particularly to the compounds I-212 and I-213which can be obtained by the process described in scheme 5. In thiscase, N-acetyl-S-trityl-L-cysteine (7) was coupled according to methodA, scheme 1: route 1) with thiazolidine to form the compound 18. TheS-detritylation of the latter according to the protocol described above,in particular for the preparation of I-152, makes it possible togenerate a free thiol known as I-212. The preparation of the compoundI-213 is carried out by S-acetylation of I-212. The three couplingproducts (18, I-212 and I-213) were respectively isolated in the form ofa mixture of two conformational isomers. These isomers are due to thepresence of the carbonyl of the pseudopeptide bond alpha to the nitrogenatom of the thiazolidine.

All the compounds of the invention exhibit a common characteristic; theyare precursors of compounds which are involved in the route for thebiosynthesis of glutathione. In other words, these compounds can be usedas intermediates which are involved in the route for the biosynthesis ofglutathione. It can relate, for example, to a product chosen from thegroup formed by NAC, MEA and L-cysteine.

The compounds of the invention exhibit an antioxidant activity. Anothersubject matter of the invention is therefore the use of the compounds asdescribed above as antioxidant agents; such compounds have a wide rangeof uses, such as use in the preventive and curative treatment ofpathological syndromes for which an oxidative stress and a GSH deficitare observed, uses in cosmetology or uses in the farm-produce industry.

The invention is targeted at providing antioxidant agents for combatingoxidative stress and for increasing the intracellular level ofglutathione. The compounds of the present invention can be used asmedicament, in particular for increasing the intracellular and/orextracellular level of glutathione. The invention also covers the use ofa compound according to the invention in the preparation of a medicamentintended to increase the intracellular and/or extracellular level ofglutathione. The present invention also relates to a pharmaceuticalcomposition, characterized in that it comprises an effective amount of acompound according to the invention and a pharmaceutically acceptablevehicle. The invention also relates to the use of a compound accordingto the invention in the preparation of a medicament or of apharmaceutical composition for the treatment and/or prevention ofpathologies or disorders related to an intra- and/or extracellulardepletion of glutathione.

The pathologies which can form the subject of a prophylaxis or of atreatment by the compounds of the invention are in particular viralinfections, bacterial infections, parasitic infections, diseases of therespiratory tract, neurodegenerative diseases, autoimmune diseases,cardiovascular diseases, cancers, diseases of the immune system,diabetes and preferably type I diabetes, ophthalmic pathologies ordermatological diseases.

The invention relates more particularly to the use of a compound asdescribed above in the preparation of a medicament or of apharmaceutical composition for the treatment and/or prevention of viralinfections; they are in particular viral infections caused by DNAviruses and RNA viruses and more particularly by retrdid viruses, moreparticularly the human immuno-deficiency virus (HIV) and preferably thetype-1 human immunodeficiency virus (HIV-1). Infection by the HIV isresponsible for acquired immunodeficiency syndrome (AIDS), whichconstitutes a human pathology in which oxidizing agents play animportant role. AIDS has constituted a public health problem in manycountries in the world since 1981, the date at which the disease wasidentified for the first time. When AIDS is declared, death generallyfollows two to three years after diagnosis as a result of the collapseof the immune defenses of the patient and of multiple opportunisticinfections. During infection by the HIV, a decrease in the cell andplasma levels of antioxidant molecules is observed. This immunedisruption, christened “oxidative stress”, is critical for the patient.It seems to play a major role in the physiopathology of infections bythe HIV by enhancing viral replication, the inflammatory syndrome,apoptosis, loss in weight of the patients (cachexia) and poisoning bymedicaments. While the mechanisms contributing to this oxidative stressare poorly understood, it seems probable that the chronic inflammatorysyndrome associated with HIV infections accentuates it. Likewise, theHIV, via the Tat protein, seems itself to play a major role. This isbecause this protein blocks the production and secretion of manganesesuperoxide dismutase (MnSOD), an enzyme which can prevent oxidativestress, and greatly decreases the activity of glucose-6-phosphatedehydrogenase (G6PD), an enzyme necessary for the maintenance ofglutathione in its reduced form.

In individuals infected by the HIV, thiols and very particularly GSH aredecreased in the plasma and the peripheral blood mononuclear cells(PBMCs). Attacks occur both in the blood and in the tissues, since theGSH deficit is found in the bronchoalveolar lavages and in the centralnervous system (CNS). These twofold localizations first confirm that thetwo major targets of HIV, the lymphocytes and the macrophages, areaffected and, secondly, illustrate the scale of the deficit. This canprobably explain, at the very least in part, the results published by L.A. Herzenberg et al. (Proc. Natl. Acad. Sci., 1997, 94, 1967-1972).These authors show the existence of a direct connection between patientsurvival and the GSH level.

Current antiretroviral therapy is based on two families of molecules,reverse transcriptase (RT) inhibitors, (AZT, ddI, nevirapine, and thelike) and viral protease inhibitors (indinavir, saquinavir, and thelike). They have a degree of in vivo activity when they are combinedwith one another. However, these molecules are incapable of fullyreorganizing affected tissues, such as, for example, those affected byinflammatory and oxidative syndromes in the CNS, and have a reduced orzero effectiveness with respect to pathologies associated with infectionby the HIV. In view of the major role of GSH in the control of these twosyndromes and of its plurality of effects in the physiopathology of HIVinfections, numerous alternatives targeted at raising its intracellularlevel have been envisaged without success as adjuvant therapeuticstrategy in recent years.

The unremitting spread of infections by the HIV virus and of theassociated opportunistic infections makes it necessary to have availablean effective treatment against AIDS and of the associated affectedtissues. One of the aims of the present invention is thus to use thecompounds corresponding to the general formula (I), preferably thecompounds I-152 and/or I-176 and/or I-177 and/or I-178, in thepreparation of a medicament or of a pharmaceutical composition for thetreatment and/or prevention of viral infections brought about by thehuman immunodeficiency virus (HIV) and more particularly the type-1human immunodeficiency virus (HIV-1). The present invention alsoprovides a pharmaceutical composition for the preventive and curativetreatment of AIDS and the associated affected tissues, characterized inthat it comprises a therapeutically effective amount of a compoundaccording to the invention and a pharmaceutically acceptable vehicle.The present invention also relates to a product comprising at least onecompound according to the invention and at least one reversetranscriptase inhibitor and/or as combination product for a use inantiviral therapy which is simultaneous, separate or spaced out overtime. The reverse transcriptase inhibitor is chosen, for example, from3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI),2′,3′-dideoxycytidine (ddC), (−)-2′,3′-dideoxy-3′-thiacytidine (3TC),2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and(−)-2′-deoxy-5-fluoro-3′-thiacytidine (FTC), TIBO, HEPT, TSAO, α-APA,nevirapine, BAHP or phosphonoformic acid (PFA). The viral proteaseinhibitor is chosen more particularly from indinavir and saquinavir.

It is also within the scope of the invention to use the compoundscorresponding to the general formula (I), preferably the compounds I-152and/or I-176 and/or I-177 and/or I-178, in the preparation of amedicament or of a pharmaceutical composition for the treatment and/orprevention of cardiovascular diseases preferably chosen from the groupconsisting of arterial hypertension, arteriosclerosis, cerebralischemia, cardiac ischemia, ventricular arrhythmias, ventricularfibrillation and myocardial infarction. This is because patientsaffected by arterial hypertension treated with organic nitratesfrequently develop resistance to the effects of these drugs. It has beensuggested that this tolerance is associated, among factors, with thedepletion of thiol groups in the vascular smooth muscles. It has beendemonstrated that metabolic precursors of glutathione, such as NAC,prevent the development of tolerance or at least restore the effects ofthe organic nitrates (Abrams 1991, Horowitz 1991, Bosegaard et al.1993). The present invention thus proposes to provide novel compoundsfor preventing the development of tolerance or at least restoring theeffects of the organic nitrates used in the treatment of arterialhypertension. Heller et al. (1997) have experimentally demonstrated theaction of various reactive oxygen species in the inflammation of theislets of Langerhans and in the destruction of the β cells. It is thusan object of the present invention to provide novel compounds for thetreatment and prevention of type-I diabetes (IDDM) (Rabinovitch et al.,1992).

Oxidative stress and GSH deficit are also involved in other pathologies.Thus, in the field of ophthalmology, they can be related to theappearance of cataracts. It is thus within the scope of the invention touse the compounds corresponding to the general formula (I), preferablythe compounds I-152 and/or I-176 and/or I-177 and/or I-178, in thepreparation of a medicament or of a pharmaceutical composition for thetreatment and/or prevention of ophthalmic pathologies, such as theocular effects of Sjogren's syndrome and cataracts.

It is also within the scope of the invention to use the compoundscorresponding to the general formula (I), preferably the compounds I-152and/or I-176 and/or I-177 and/or I-178, in the preparation of amedicament or of a pharmaceutical composition for the treatment and/orprevention of diseases of the respiratory tract, in particular pulmonaryemphysema, idiopathic pulmonary fibrosis, cystic fibrosis, chronicbronchitis, acute bronchitis or adult respiratory distress syndrome.

The invention also relates to the use of a compound as described abovein the preparation of medicaments intended for the preventive and/orcurative treatment of noise-related hearing loss.

The invention also relates to the use of a compound as described abovein the preparation of medicaments intended for the treatment ofpoisoning related to the oral or parenteral administration, as or not asan overdose, of substances preferably chosen from the group consistingof acetaminophen, nitrites, ethanol, acrylonitrile and heavy metals,more particularly gold, silver and mercury.

The antioxidant properties of the compound of the invention recommend itfor use in the field of cosmetics. This is because antioxidants arealready used in cosmetology to slow down aging. The compounds of thepresent invention are capable of promoting the rebuilding of the cellcontent of GSH and of providing effective protection against cell damagecaused by extrinsic and intrinsic toxic factors; the skin is the site ofattack by these factors. The extrinsic factors include, for example,ultraviolet radiation, wind, low humidity, abrasives and strongsurface-active agents. The intrinsic factors include chronological agingand biochemical modifications of the skin. Whether extrinsic orintrinsic, these factors lead to the appearance of wrinkles and otherhistological changes associated with aging of the skin. The antiwrinkleagents currently known include compounds such as N-acetyl-L-cysteine,retinoids, such as retinoic acid, and alpha-hydroxy acids, such asglycolic acid and lactic acid. It is thus one of the objects of theinvention to use the antioxidant properties of the compounds accordingto the invention to (i) prevent, erase and treat wrinkles or fine linesof the skin; and/or (ii) combat cutaneous and/or subcutaneousslackening; and/or (iii) improve the texture of the skin and rekindlethe radiance of the skin; and/or (iv) remove undesired hairs from theskin; and/or (v) decrease the sizes of the pores of the skin; and/or(vi) permanently deform the hair. As regards the final point, it shouldbe pointed out that organic molecules carrying thiol functional groups,such as the compounds according to the invention, are products havingmultiple applications. One of these applications is the permanentdeformation (curling and straightening) of the hair, which consists, ina first step, in opening the disulfide bonds (S—S) of the cystine unitsof the keratin using a composition comprising at least one organiccompound carrying a thiol functional group which acts as reducing agent(reduction stage), which makes it possible to confer the desired shapeon the hair; then, after having rinsed the hair, in reconstituting, in asecond step, said disulfide bonds by applying, to the hair, an oxidizingcomposition (oxidation or fixation stage), so as to fix the hair in theshape which has been given to it. The invention thus relates to acosmetic composition for the treatment of the skin and/or hair and/orbody hairs, characterized in that it comprises a compound according tothe invention and a cosmetically acceptable excipient. The inventionalso relates to a process for the cosmetic treatment of the skin forpreventing, erasing and treating wrinkles or fine lines of the skinand/or combating cutaneous and/or subcutaneous slackening and/orimproving the texture of the skin and rekindling the radiance of theskin and/or removing undesired hairs from the skin and/or decreasing thesizes of the pores of the skin which comprises the application, to theskin, of a cosmetic composition as described above.

The antioxidant properties of the compound of the invention recommend itfor use in the farm-produce field. It is thus within the scope of thisinvention to use the compounds of the invention as antioxidant agentsfor the preservation of the organoleptic and nutritional properties ofdrinks, in particular fruit juices, and food.

Other characteristics and advantages of the present invention will bemore clearly demonstrated on reading the following examples: in theseexamples, reference will be made to the following figures:

FIG. 1: Probable decomposition of I-152.

FIG. 2: Comparison of the antiviral activities of NAC, of MEA and ofI-152 in MDM cultures infected with 10 000 TCID50s of the HIV-1/Ba-Lisolate: effect-doses. The results are expressed according to themean±standard deviation of the percentages of inhibition. Viralreplication was measured by assaying the reverse transcriptase activityin the culture supernatants.

FIG. 3: Cytotoxicity of NAC, MEA and I-152 with respect to MDMs.

FIG. 4: Measurement of the RT activity and of the production of proteinp25 in the MDM culture supernatants infected with the HIV-1/Ba-L strainand treated with I-152.

FIG. 5: Effect of the m.o.i. on the antiviral activity of I-152. TheMDMs were infected with 1 000 or 10 000 TCID50s of the HIV-1/Ba-Lisolate.

FIG. 6: Effects of I-152 on viral replication according to the treatmentmethod: pretreatment 24 hours, treatment 24 hours after infection,treatment 7 days after infection.

FIG. 7: Antiviral activity of I-152 in quiescent PBMCs or BMCs activatedwith PHA-P, and infected with the HIV-1 LAI strain.

FIG. 8: Antiviral activity of I-152 in quiescent PBLs or PBLs activatedwith PHA-P, then infected with the HIV-1 LAI strain.

FIG. 9: Effects of I-152 on the integration of the provirus within thecell genome.

FIG. 10: Effects of I-152 on the enzymatic activity of the RT of theHIV-1. The experiments were carried out in triplicate.

FIG. 11: Assaying of the total intracellular glutathione in quiescentPBMCs, treated 24 hours before with NAC, MEA or I-152.

FIG. 12: Antiviral activity of the I-152 derivatives (I-176 iscytotoxicat the dose tested).

FIG. 13: Intracellular concentration of GSH in MDMs infected or notinfected, in vitro with the reference strain with macrophage tropismHIV-1/Ba-L, and treated or not treated with the compound I-152.

FIG. 14: Effects of the compound I-152 on the intracellularconcentration of GSH and the synthesis of TNFα in MDMs stimulated invitro with a bacterial lipopolysaccharide (LPS; 1 μg/ml) and IFN-γ (100IU/ml).

FIG. 15: Intracellular concentration of GSH in spleen macrophages afteror not after treatment with I-152. The intracellular concentration ofGSH in untreated human spleen macrophages is 22±2 μM.

FIG. 16: Intracellular concentration of GSH in spleen macrophagesinfected or not infected in vitro with the reference strain withmacrophage tropism HIV-1/Ba-L.

FIG. 17: Effects of I-152 on the anti-HIV activity of AZT in MDMsinfected with the HIV-1/Ba-L strain.

FIG. 18: Comparison of the antiviral activities of NAC, MEA and I-152 inMDM cultures infected with 10 000 TCID50s of the HIV-1/Ba-L isolate: 50,70 and 90% effective doses.

FIG. 19: Effects of the m.o.i. on the antiviral activity of I-152: 50%effective doses.

FIG. 20: Antiviral activity of I-152 in spleen MDMs infected with 10 000TCID50s of the HIV-1/Ba-L isolate: 50, 70 and 90% effective doses.

FIG. 21: Comparison of the antiviral activities of I-152 and itsS-acylated analogues in MDM cultures infected with 10 000 TCID50s of theHIV-1/Ba-L isolate: 50, 70 and 90% effective doses.

FIG. 22: Comparison of the antiviral activities of I-152 and itsvariously S-acylated (N-isobutyryl) derivatives in MDM cultures infectedwith 10 000 TCID50s of the HIV-1/Ba-L isolate: 50, 70 and 90% effectivedoses.

EXAMPLE 1 Synthesis of N-(N-Acetyl-L-cysteinyl)-S-acetylcysteamine(I-152)

1.1 First Route for the Synthesis of I-152 Using S,N-ProtectedL-Cysteine:

1.1.1. N-(N-Acetyl-S-trityl-L-cysteinyl)-S-acetylcysteamine (8)

a)—Coupling Method Involving in situ a Mixed Anhydride

A solution comprising 290 mg (0.71 mmol) of N-acetyl-S-trityl-L-cysteine(7, Bachem) and 80 μl (0.72 mmol) of of N-methylmorpholine (NMM) in 5 mlof AcOEt is stirred at −15° C. and then 93 μl (0.71 mmol) of isobutylchloroformate are added. After stirring for 15 min and while maintainingthe starting temperature, 111.4 mg (0.71 mmol) of S-acetylcysteaminehydrochloride (prepared according to T. Wieland and E. Bokelman, Ann.Chem., 1952, 576, 20-34) and then 80 μl (0.72 mmol) of NMM are added.The reaction mixture is maintained at −15° C. for 15 min and then, afterreturning to room temperature, stirring is continued for 3 h. The NMMhydrochloride formed is filtered off and washed with 2×2.5 ml of AcOEtand the combined organic phases are evaporated to dryness under vacuum.The coupling product is subsequently isolated from the gum obtained byflash chromatography on a silica gel column (eluent: AcOEt/petroleumether 30%). 198 mg (Yd=55%) of the expected compound are collected.R_(f) (AcOEt/petroleum ether, 9:1): 0.41. Cystallizes from anAcOEt/petroleum ether mixture in the form of a colorless powder:M.p.=111-113° C. [α]_(D) ²⁰=+10.5° (c 0.8, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.90 (s, 3H, NCOCH₃), 2.29 (s, 3H, SCOCH₃), 2.48(dd, J=5.7, 12.9 Hz, 1H, β Ha cys), 2.82 (dd, J=6.4, 12.9 Hz, 1H, β Hbcys), 2.92-3.01 (m, 2H, NCH₂CH₂S) 3.32-3.42 (m, 2H, NCH₂CH₂S), 4.07-4.20(m, 1H, α H cys), 5.70 (d, J=7.6 Hz, 1H, NH cys), 6.34 (t, J=5.5 Hz, 1H,NHCH₂), 7.19-7.35 and 7.40-7.47 (2m, 15H, aromatic H). MS: (FAB⁺/NBA+K⁺)m/z 545 (M+K)⁺, 507 (M+H)⁺; (FAB⁻/NRA) m/z 505 (M−H)⁻.

Analysis: C₂₈H₃₀N₂O₃S₂ (506) Calc. %: C 66.40 H 5.93 N 5.53 Found %:66.17 6.00 5.81b)—Coupling Method Involving in situ an Activated Ester

A solution comprising 1.5 g (3.70 mmol) of 7 and 410 μM (3.73 mmol) ofNMM in 30 ml of AcOEt is stirred at −15° C. and then 480 μl (3.70 mmol)of isobutyl chloroformate are added. After stirring for 15 min and whilemaintaining the starting temperature, 426 mg (3.70 mmol) ofN-hydroxysuccinimide are added. The reaction mixture is maintained at−15° C. for 15 min and then, after returning to ambient temperature,stirring is continued for 2 h. The NMM hydrochloride formed is filteredoff and washed with 2×5 ml of AcOEt. The organic phases comprising theO—N-succinimide active ester of 7 are combined and stirred at −15° C.575 mg (3.70 mmol) of S-acetylcysteamine hydrochloride and 410 μl (3.73mmol) of NMM are then successively added to the solution. The reactionmixture is maintained at −15° C. for 15 min and then, after returning toambient temperature, stirring is continued for 12 h. The solution issubsequently diluted with 300 ml of AcOEt, washed (water, 30 ml;ice-cold saturated sodium bicarbonate, 30 ml; water, 30 ml; ice-cold0.1N citric acid, 30 ml; water, 3×30 ml), dried over sodium sulfate,filtered and evaporated to dryness under vacuum. The residue obtained issubsequently purified as above to give, with a yield of 70% (1.31 g) andwith entirely the same physicochemical criteria, the coupling product 8described above.

1.1.2. N-(N-Acetyl-L-cysteinyl)-S-acetylcysteamine (I-152)

A saturated solution, with the exclusion of light, of 1.26 g (2.49 mmol)of 8 in 20 ml of MeOH and 1.5 ml of CHCl₃ is stirred at ambienttemperature and a mixture, also with the exclusion of light, comprising449 mg (2.64 mmol) of silver nitrate and 213 μl (2.64 mmol) of pyridinein 13 ml of MeOH, is added. There is instantaneously formation of aprecipitate of the corresponding silver sulfide 9. At the end of theaddition, stirring is halted and the reaction mixture is left overnightat ambient temperature. The precipitate is subsequently filtered off andwashed with MeOH (2×10 ml) and then with CHCl₃ (2×10 ml).

An analytical sample of 9 is withdrawn and is then dried under vacuumwith the exclusion of light.

Analysis: C₉H₁₅N₂O₃S₂Ag (371) Calc. %: Ag 29.11 Found %: 29.16

The preceding sulfide 9 is suspended in 15 ml of CHCl₃ and stirred atambient temperature with the exclusion of light and then 400 μl ofconcentrated hydrochloric acid are added. Stirring is continued for 2 hat ambient temperature and then for 2 min at 30-35° C. The mixture isthen diluted with 70 ml of CHCl₃ and the silver chloride formed isfiltered off and then washed with 3×10 ml of the same solvent. Theorganic phases are combined, rapidly washed with ice-cold water (3×10ml), dried over sodium sulfate, filtered and evaporated to dryness undervacuum. A semi-crystalline paste is collected, which paste crystallizesfrom an AcOEt/petroleum ether mixture in the form of colorlessmicrocrystals (368 mg, Yd=56%). M.p.=121-122° C. [α]_(D) ²⁰=−39.1° (c0.9, CHCl₃). The other data (microanalyses and spectra) are in allrespects identical to those described in the second synthetic route.

H₂S was also used and leads to the same result.

1.2. Second Route for the Synthesis of I-152 Using N-Protected L-Serine:

1.2.1. N-(N-Boc-L-Seryl)-2-aminoethanol (3)

A solution comprising 6.15 g (30 mmol) of N-Boc-L-serine (1, Fluka) and3.45 g of N-hydroxysuccinimide (30 mmol) in 80 ml of DMF is stirred at0° C. and 6.2 g (30 mmol) of DCC are added. The reaction mixture ismaintained at 0° C. for 15 min, then it is allowed to return to ambienttemperature and stirring is continued for 1 h 30. 2.75 ml (60 mmol) ofethanolamine are subsequently added. After stirring for 12 h, the DCUformed is filtered off and washed with 2×15 ml of DMF. The combinedorganic phases are evaporated to dryness under vacuum. The product isisolated from the residual paste by flash chromatography on a silica gelcolumn (Kieselgel Merck 60, 230-400 mesh; eluent: CH₂Cl₂/MeOH 6%). Theexpected compound is collected in the form of a gum which crystallizesfrom an AcOEt/hexane mixture to provide 5.21 g (Yd=70%) of colorlessneedles. R_(f) (CH₂Cl₂/MeOH, 9.3/0.7): 0.23; (CH₂Cl₂/MeOH/AcOH,9/0.9/0.1): 0.47. M.p.=74-76° C. [α]_(D) ²⁰=−2.2° (c 0.9, CHCl₃).

¹H NMR (d₆-DMSO) δ ppm: 1.50 (s, 9H, H t-butyl), 3.18-3.29 (m, 2H,NCH₂CH₂O), 3.45-3.55 (m, 2H, NCH₂CH₂O), 3.57-3.70 (m, 2H, β CH₂ ser),4.01-4.10 (m, 1H, α H ser), 4.77 (t, J=5.4 Hz, 1H, OH ser), 4.91 (t,J=5.7 Hz, 1H, NCH₂CH₂OH), 6.71 (d, J=8.2 Hz, 1H, NH ser); 7.86 (t, J=5.5Hz, 1H, NHCH₂). MS: (FAB⁺/G-T) m/z 745 (3M+H)⁺, 497 (2M+H)⁺, 249 (M+H)⁺.

Analysis: C₁₀H₂₀N₂O₅ (248) Calc. %: C 48.39 H 8.06 N 11.29 Found %: 8.57 8.08 11.08

1.2.2. N-(N-Boc-S-Acetyl-L-cysteinyl)-2-acetylcysteamine (4)

A solution comprising 2.597 g (9.9 mmol) of triphenylphosphine and 1.95ml (9.91 mmol) of diusopropyl azodicarboxylate in 15 ml of THF isstirred for 30 min at 0° C. (after stirring for 30 s, the formation ofan intense precipitate is observed). While retaining this temperature,1.116 g (4.50 mmol) of 3, in solution in 6 ml of THF, and then 707 μl(9.91 mmol) of thioacetic acid are successively added. The solutionobtained is subsequently allowed to return to ambient temperature andstirring is continued for 12 h. The reaction mixture is then evaporatedto dryness under vacuum. The product is isolated from the residual pasteby flash chromatography on a silica gel column (eluent: hexane and thenAcOEt/petroleum ether 75%). The expected compound is collected in theform of a gum which crystallizes from an AcOEt/petroleum ether mixturein the form of colorless needles (1.21 g, Yd=74%). R_(f)(AcOEt/petroleum ether, 4/6): 0.35. M.p.=111-113° C. [α]_(D) ²⁰=−13.9°(c 0.86, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.45 (s, 9H, H t-butyl), 2.36, 2.38 (2s, 2×3H,2×SCOCH₃), 2.99-3.07 (m, 2H, NCH₂CH₂S), 3.19 (dd, J=7.8, 14.3 Hz, 1H, βHa cys), 3.34 (dd, J=4.5, 14.3 Hz, 1H, β Hb cys), 3.40-3.50 (m, 2H,NCH₂CH₂S), 4.19-4.34 (m, 1H, α H cys), 5.25 (d, J=7.1 Hz, 1H, NH cys),6.69 (t, J=5.2 Hz, 1H, NHCH₂). MS: (FAB⁺/NBA) m/z 729 (2M+H)⁺, 365(M+H)⁺.

Analysis: C₁₄H₂₄N₂O₅S₂ (364) Calc. %: C 46.15 H 6.59 N 7.69 Found %:46.45 6.51 7.47

1.2.3. N-(N-Acetyl-L-cysteinyl)-S-Acetylcysteamine (I-152)

A solution comprising 500 mg (1.37 mmol) of 4 in 5 ml of CH₂Cl₂ isstirred under argon at 0° C. and then 1 ml (13.07 mmol) of TFA is added.The solution is subsequently allowed to return to ambient temperatureand stirring is continued for 7 h. At this stage, the reaction,monitored by TLC(CH₂Cl₂/MeOH, 9.4/0.6), shows the disappearance of thestarting compound (R_(f): 0.75) and the appearance of two, more polar,spots (R_(f): 0.5 and 0.16). The reaction mixture is evaporated todryness under vacuum (temperature of the water bath: <40° C.).

Further monitoring by TLC of the gum obtained indicates that thepreceding major spot at R_(f): 0.16 has become a minor spot to theadvantage of that at R_(f): 0.5. The appearance of a third spot, of lessimportance, at R_(f): 0.4 is also recorded. At this stage, we carriedout flash chromatography on an aliquot of the gum (20 mg) in order toidentify the compounds present in order to elucidate this phenomenon:

-   -   The product of R_(f): 0.5 is isolated with a mixture of eluents        consisting of CH₂Cl₂/Et₂O (5/5). The study of its spectra (¹H        NMR and MS) unambiguously shows that it is        N-(2-methyl-Δ²-thiazolinyl-4(R)-carbonyl)-S-acetylcysteamine 6:

¹H NMR (CDCl₃) δ ppm: 2.32 (s, 3H, SCOCH₃), 2.39 (d, J=1.3 Hz, 3H, 2—CH₃thiazoline), 3.05 (app. t, J=6.4 Hz, 2H, NCH₂CH₂S), 3.36-3.60 (m, 2H,NCH₂CH₂S), 3.69 (dd, J=10.2 and 11.4 Hz, 1H, 5-H thiazoline), 3.85 (dd,J=7.2 and 11.4 Hz, 1H, 5′-H thiazoline), 5.08 (ddd, J=1.3, 7.2 and 10.2Hz, 1H, 4-H thiazoline), 7.45-7.58 (m, 1H, NHCH₂). MS: (FAB⁺/G-T) m/z493 (2M+H)⁺, 247 (M+H)⁺.

-   -   The intermediate compound (R_(f): 0.4) is isolated by increasing        the polarity of the elution solvent (CH₂Cl₂/MeOH, 9.5/0.5). The        study of its spectra (¹H NMR and MS) shows that it is I-152. Its        physicochemical data are reported at the end of this        description.    -   Continuing the chromatographic separation with more polar        eluents (CH₂Cl₂/MeOH 5-20%) did not make it possible to obtain        the product of R_(f): 0.16. This compound, which is the first        formed in the reaction for the deprotection of the terminal        amine functional group of the starting N-Boc, can only be        N-(S-acetyl-L-cysteinyl)-S-acetylcysteamine trifluoroacetate 5.        * All the tests carried out and monitored by TLC showed us,        under our operating conditions (time necessary for the complete        consumption of the starting material, temperature and pH of the        reaction medium), that 5, which is the first formed during        deprotection of the N-Boc by TFA, generates the cyclic        intermediate 6, which is subsequently slowly hydrolyzed to give        I-152.

Thus, after these various observations, the remaining gum obtained afterevaporation of the crude reaction mixture was dissolved in 150 ml ofCH₂Cl₂, and then 5 ml of water were added at ambient temperature andwith vigorous stirring. After stirring for 6 h, monitoring by TLC showsonly a single spot corresponding to the desired product. The organicphase is separated by settling and the residual water is extracted withCH₂Cl₂ (3×10 ml). The organic phases are subsequently combined, driedover sodium sulfate, filtered and evaporated to dryness under vacuum.The expected compound is collected in the form of a semi-crystallinepaste. R_(f) (CH₂Cl₂/MeOH, 9.5/0.5): 0.4. Crystallizes from anAcOEt/petroleum ether mixture in the form of colorless microcrystals(245 mg, Yd=≧67%). M.p.=122-124° C. [α]_(D) ²⁰=−40° (c 0.87, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.60 (dd, J=7.6 and 10.3 Hz, 1H, SH), 2.07 (s, 3H,NCOCH₃), 2.36 (s, 3H, SCOCH₃), 2.70 (ddd, J=6.5, 10.3 and 13.9 Hz, 1H, βHa cys), 3.03 (t, J=6.3 Hz, 2H, NCH₂CH₂S), 3.06 (ddd, J=4.3, 7.6 and13.9 Hz, 1H, β Hb cys), 3.46 (td, J=6.0 and 6.3 Hz, 2H, NCH₂CH₂S), 4.59(ddd, J=4.3, 6.5 and 7.9 Hz, 1H, α H cys), 6.52 (d, J=7.9 Hz, 1H, NHcys), 6.75-6.90 (m, 1H, NHCH₂). MS: (FAB⁺/G-T) m/z 529 (2M+H)⁺, 265(M+H)⁺.

Analysis: C₉H₁₆N₂O₃S₂ ₍₂₆₄₎ Calc. %: C 40.91 H 6.06 N 10.61 S 24.24Found %: 41.21 6.00 10.91 23.99

EXAMPLE 2 Synthesis of Acylated Derivatives ofN-(N-Acetyl-L-cysteinyl)-S-acetylcysteamine (I-152) or of ItsDerivatives

2.1. N-(N,S-Bisacetyl-L-cysteinyl)-S-acetylcysteamine I-176)

(General Method for S-Acylation)

A solution comprising 83 mg (0.31 mmol) of I-152 in 1 ml of pyridine isstirred at 0° C. and 90 μm (0.95 mmol) of acetic anhydride are added.The reaction mixture is maintained at 0° C. for 15 min, then it isallowed to return to ambient temperature and stirring is continued for12 h. The solution is subsequently evaporated to dryness under vacuumand the residue formed is taken up in 30 ml of CH₂Cl₂. The organic phaseis washed with water (3×20 ml), dried over sodium sulfate, filtered andevaporated to dryness under vacuum. The residual gum is crystallizedfrom AcOEt and provides 73 mg (Yd=75%) of the expected compound in theform of colorless platelets. R_(f) (CH₂Cl₂/MeOH, 9.5/0.5): 0.46.M.p.=153-154° C. [α]_(D) ²⁰=−33.7° (c 0.8, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 2.02 (s, 3H, NCOCH₃), 2.37, 2.39 (2s, 2×3H,2×SCOCH₃), 2.93, 3.12 (m, 2H, NCH₂CH₂S), 3.24 (dd, J=7.2 and 14.5 Hz,1H, β Ha cys), 3.31 (dd, J=5.2 and 14.5 Hz, 1H) β Hb cys), 3.39-3.49 (m,2H, NCH₂CH₂S), 4.54 (ddd, J=5.3, 7.2 and 7.3 Hz, 1H, α H cys), 6.44 (d,J=7.3 Hz, 1H, NH cys), 6.83 (t, J=5.1 Hz, 1H, NHCH₂). MS: (FAB⁺/G-T) m/z613 (2M+H)⁺, 307 (M+H)⁺.

Analysis: C₁₁H₁₈N₂O₄S₂ (306) Calc. %: C 43.14 H 5.88 N 9.15 S 20.92Found %: 42.95 5.96 8.93 20.642.2. N-(N-Acetyl-S-isoutyryl-L-cysteiyl-S-acetylcysteamine (I-177)

The acylation reaction of 85 mg (0.32 mmol) of I-152 is carried out,according to the general method described above, using 137 μl (1.30mmol) of isobutyryl chloride instead of acetic anhydride. The viscousmixture obtained, after evaporation to dryness under vacuum, issubsequently diluted with 30 ml of CH₂Cl₂. The solution is subsequentlywashed (water, 10 ml; ice-cold saturated sodium bicarbonate, 10 ml;water, 10 ml; ice-cold 0.1N citric acid, 10 ml; water, 3×10 ml), driedover sodium sulfate, filtered and evaporated to dryness under vacuum.The acylation product is isolated from the gum obtained by flashchromatography on a silica gel column (eluent: CH₂Cl₂/ether 50%). 60 mg(Yd=56%) of the expected compound are collected. R_(f) (CH₂Cl₂/MeOH,9.4/0.6): 0.6. Crystallizes from an AcOEt/petroleum ether mixture in theform of colorless platelets. M.p.=116-118° C. [α]_(D) ²⁰=−18.4° (c 0.87,CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.20 (d, J=6.9 Hz, 6H, C(CH₃)₂, 2.00 (s, 3H,NCOCH₃), 2.37 (s, 3H, SCOCH₃), 2.80 (hept, J=6.9 Hz, 1H, CH(CH₃)₂, 2.93,3.12 (m, 2H, NCH₂CH₂S), 3.23-3.30 (m, 2H, β CH₂ cys), 3.38-3.49 (m, 2H,NCH₂CH₂S), 4.46-4.57 (m, 1H, α H cys), 6.42 (d, J=7.3 Hz, 1H, NH cys),6.80 (t, J=5.2 Hz, 1H, NHCH₂). MS: (FAB⁺/G-T) m/z 669 (2M+H)⁺, 335(M+H)⁺.

Analysis: C₁₃H₂₂N₂O₄S₂ (334) Calc. %: C 46.71 H 6.59 N 8.38 S 19.16Found %: 46.76 6.89 8.24 19.322.3. N-(N-Acetyl-S-pivaloyl-L-cysteinyl)-S-acetylcyste-amine (I-178)

The acylation reaction of 95 mg (0.36 mmol) of I-152 is carried out,according to the general method described above, using 176 μl (1.44mmol) of pivaloyl chloride instead of acetic anhydride. The viscousmixture obtained, after evaporation to dryness under vacuum, issubsequently diluted with 30 ml of CH₂Cl₂. The solution is subsequentlywashed (water, 10 ml; ice-cold saturated sodium bicarbonate, 10 ml;water, 10 ml; ice-cold 0.1N citric acid, 10 ml; water, 3×10 ml), driedover sodium sulfate, filtered and evaporated to dryness under vacuum.The acylation product is isolated from the gum obtained by flashchromatography on a silica gel column (eluent: CH₂Cl₂/ether 50%). 70 mg(Yd=56%) of the expected compound are collected. R_(f) (CH₂Cl₂/ether,4/6):0.23. Crystallizes from an AcOEt/petroleum ether mixture in theform of colorless platelets. M.p.=92-94° C. [α]_(D) ²⁰=−11.1° (c 1.08,CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.25 (s, 9H, C(CH₃)₃, 2.00 (s, 3H, NCOCH₃), 2.37(s, 3H, SCOCH₃), 2.94, 3.12 (m, 2H, NCH₂CH₂S), 3.25 (d, J=6.5 Hz, 2H, βCH₂ cys), 3.38-3.49 (m, 2H, NCH₂CH₂S); 4.51 (td, J=6.5 and 7.3 Hz, 1H, αH cys), 6.42 (d, J=7.3 Hz, 1H, NH cys), 6.80 (t, J=5.2 Hz, 1H, NHCH₂).MS: (FAB⁺/G-T) m/z 697 (2M+H)⁺, 349 (M+H)⁺.

Analysis: C₁₄H₂₄N₂O₄S₂ (348) Calc. %: C 48.28 H 6.90 N 8.05 S 18.39Found %: 48.32 6.95 7.94 18.432.4 N-(N-Acetyl-S-trityl-L-cysteinyl)-S-isobutyrylcysteamine (10)

The coupling reaction of 7 (4.5 mmol) with S-isobutyrylcysteaminehydrochloride [(compound obtained according to the same methods as thosedescribed by T. Wieland and E. Bokelman, Ann. Chem., 1952, 576; 20-34;during the syntheses of S-acetylcysteamine hydrochloride andS-benzoylcysteamine hydrochloride) M.p.=147-148° C.] is carried outaccording to method B described in the first synthetic route (example1). After the various treatments, the expected compound is isolated byflash chromatography on a silica gel column (eluent: CH₂Cl₂/ether 30%).10 is collected in the form of a colorless foam with a yield of 80%.R_(f) (AcOEt/petroleum ether, 8/2): 0.37. [α]_(D) ²⁰=+10° (c 1.1,CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.16 (d, J=6.9 Hz, 6H, C(CH₃)₂), 1.90 (s, 3H,NCOCH₃), 2.49 (dd, J=5.7 and 12.9 Hz, 1H, β Ha cys), 2.70 (hept, J=6.9Hz, 1H, CH(CH₃)₂), 2.79 (dd, J=6.4 and 12.9 Hz, 1H, β Hb cys), 2.88-3.01(m, 2H, NCH₂CH₂S), 3.29-3.41 (m, 2H, NCH₂CH₂S), 4.08-4.19 (m, 1H, α Hcys), 5.76 (d, J=7.7 Hz, 1H, NH cys), 6.36 (t, J=5.5 Hz, 1H, NHCH₂),7.15-7.35, 7.38-7.52 (2m, 15H, aromatic H). MS: (FAB⁺/G-T)m/z 535(M+H)⁺.

Analysis: C₃₀H₃₄N₂O₃S₂ (534) Calc. %: C 67.42 H 6.37 N 5.24 Found %:67.25 6.58 5.302.5. N-(N-Acetyl-L-cysteinyl)-S-isobutyrylcysteamine (I-188)

This compound is obtained by, the S-detritylation of 10 (2.38 mmol). Theprotocol used is the same as that described in example 1 for thesynthesis of I-152. After the various treatments, a gum is collected,which gum is purified by flash chromatography on a silica gel column(eluent: CH₂Cl₂/MeOH 1.5%). I-188 is isolated in the form of a colorlesssemicrystalline gum with a yield of 57%.

R_(f) (CH₂Cl₂/MeOH, 9.5/0.5): 0.45. [α]_(D) ²⁰=−26.6° (c 1.09, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.21 (d, J=6.9 Hz, 6H, C(CH₃)₂), 1.61 (dd, J=7.6and 10.3 Hz, 1H, SH), 2.09 (s, 3H, NCOCH₃), 2.70 (ddd, J=6.4, 10.3 and13.8 Hz, 1H, β Ha cys), 2.77 (hept, J=6.9 Hz, 1H, CH(CH₃)₂), 2.99-3.08(m, 2H, NCH₂CH₂S), 3.09 (ddd, J=4.1, 7.6 and 13.8 Hz, 1H, β Hb cys),3.41-3.53 (m, 2H, NCH₂CH₂S), 4.60 (ddd, J=4.1, 6.4 and 7.5 Hz, 1H, α Hcys), 6.48 (d, J=7.5 Hz, 1H, NH cys), 6.68-6.88 (m, 1H, NHCH₂). MS:(FAB⁺/G-T) m/z 585 (2M+H)⁺, 293 (M+H)⁺.

Analysis: C₁₁H₂₀N₂O₃S₂ (292) Calc. %: C 45.20 H 6.85 N 9.59 Found %:45.31 7.09 9.412.6. N-(N,S-Bisacetyl-L-cysteinyl)-S-isobutyrylcysteamine (I-189)

The S-acylation of I-188 (0.32 mmol) with acetic anhydride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated to the protocol described for thesynthesis of I-177. After the various treatments, a gum is collected,which gum is purified by flash chromatography on a silica gel column(eluent: CH₂Cl₂/ether 55%). I-189 is isolated in the form of a gum(Yd=64%) which, after trituration in hexane, provides a colorlesspowder. R_(f) (CH₂Cl₂/MeOH, 9.5/0.5): 0.58. M.p.=115-117° C. [α]_(D)²⁰=−20.2° (c 1.04, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.20 (d, J=6.9 Hz, 6H, C(CH₃)₂), 2.02 (s, 3H,NCOCH₃), 2.38 (s, 3H, SCOCH₃), 2.78 (hept, J=6.9 Hz, 1H, CH(CH₃)₂),2.96-3.06 (m, 2H, NCH₂CH₂S), 3.19-3.36 (m, 2H, CH₂ cys), 3.38-3.48 (m,2H, NCH₂CH₂S), 4.47-4.60 (m, 1H, α H cys), 6.37 (d, J=7.1 Hz, 1H, NHcys); 6.70-6.83 (m, 1H, NHCH₂). MS: (FAB⁺/G-T) m/z 669 (2M+H)⁺, 335(M+H)⁺.

Analysis: C₁₃H₂₂N₂O₄S₂ (334) Calc. %: C 46.71 H 6.59 N 8.38 Found %:46.81 6.62 8.362.7. N-(N-Acetyl-S-isobutyryl-L-cysteinyl)-S-isobutyrylcysteamine(I-190)

The S-acylation of I-188 (0.32 mmol) with isobutyryl chloride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum is purified by flash chromatography on a silica gelcolumn (eluent: CH₂Cl₂/ether 50%). I-190 is isolated in the form of agum (Yd=63%) which, after trituration in hexane, provides a colorlesspowder. R_(f) (CH₂Cl₂/ether, 3.5/6.5): 0.34. M.p.=99-100° C. [α]_(D)²⁰=−9.1° (c 0.88, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.20 (d, J=6.9 Hz, 12H, C(CH₃)₂), 2.01 (s, 3H,NCOCH₃), 2.78 (hept, J=6.9 Hz, 2H, CH(CH₃)₂), 2.92-3.10 (m, 2H,NCH₂CH₂S), 3.26 (d, J=6.4 Hz, 2H, CH₂ cys), 3.37-3.48 (m, 2H, NCH₂CH₂S),4.46-4.58 (m, 1H, α H cys), 6.38 (d, J=7.3 Hz, 1H, NH cys), 6.73-6.83(m, 1H, NHCH₂). MS: (FAB⁺/G-T) m/z 725 (2M+H)⁺, 363 (M+H)⁺.

Analysis: C₁₅H₂₆N₂O₄S₂ (362) Calc. %: C 49.72 H 7.18 N 7.73 Found %:49.57 7.19 7.682.8. N-(N-Acetyl-S-pivaloyl-L-cysteinyl)-S-isobutyrylcysteamine (I-191)

The S-acylation of I-188 (0.32 mmol) with isobutyryl chloride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum crystallizes from an AcOEt/petroleum ether mixturein colorless needles (Yd=68%). R_(f) (CH₂Cl₂/ether, 5/5): 0.27.M.p.=103-104° C. [α]_(D) ²⁰=−8.3° (c 0.97, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.20 (d, J=6.9 Hz, 6H, C(CH₃)₂, 1.25 (s, 9H,C(CH₃)₃), 2.01 (s, 3H, NCOCH₃), 2.77 (hept, J=6.9 Hz, 1H, CH(CH₃)₂),2.94-3.08 (m, 2H, NCH₂CH₂S), 3.25 (d, J=6.4 Hz, 2H, CH₂ cys), 3.37-3.49(m, 2H, NCH₂CH₂S), 4.44-4.57 (m, 1H, α H cys), 6.35 (d, J=7.3 Hz, 1H, NHcys), 6.69-6.80 (m, 1H, NHCH₂). MS: (FAB⁺/G-T) m/z 753 (2M+H)⁺, 377(M+H)⁺.

Analysis: C₁₆H₂₈N₂O₄S₂ (376) Calc. %: C 51.06 H 7.45 N 7.45 Found %:51.16 7.53 7.442.9. N-(N-Acetyl-S-benzoyl-L-cysteinyl)-S-isobutyrylcysteamine (I-192)

The S-acylation of I-188 (0.32 mmol) with benzoyl chloride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis I-177. After the various treatments, a gum is collected,which gum is purified by flash chromatography on a silica gel column(eluent: CH₂Cl₂/ether 35%). I-192 is isolated in the form of a gum(Yd=63%) which, after trituration in hexane, provides a colorlesspowder. R_(f) (CH₂Cl₂/ether, 5/5): 0.27. M.p.=137-138° C. [α]_(D)²⁰=+2.8° (c 1.08, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.18 (d, J=6.9 Hz, 6H, C(CH₃)₂), 2.02 (s, 3H,NCOCH₃), 2.73 (hept, J=6.9 Hz, 1H, CH(CH₃)₂), 2.96-3.06 (m, 2H,NCH₂CH₂S), 3.39-3.49 (m, 2H, NCH₂CH₂S), 3.50 (d, J=6.2 Hz, 2H, CH₂ cys),4.60-4.72 (m, 1H, α H cys), 6.59 (d, J=7.3 Hz, 1H, NH cys), 6.85-6.97(m, 1H, NHCH₂), 7.41-7.52, 7.57-7.65 and 7.93-8.01 (3m, 5H, aromatic H).MS: (FAB⁺/G-T) m/z 793 (2M+H)⁺, 397 (M+H)⁺.

Analysis: C₁₈H₂₄N₂O₄S₂ (396) Calc. %: C 54.55 H 6.06 N 7.07 Found %:54.89 6.11 7.062.10. N-(N-Acetyl-S-trityl-L-cysteinyl)-S-pivaloylcysteamine (11)

The coupling reaction of 7 (7.4 mmol) with S-pivaloylcysteaminehydrochloride [(compound obtained according to the same methods as thosedescribed by T. Wieland and E. Bokelman, Ann. Chem., 1952, 576, 20-34,during the syntheses of S-acetylcysteamine hydrochloride andS-benzoylcysteamine hydrochloride) M.p.=212-213° C.] is carried outaccording to method B described in the first synthetic route (example1). After the various treatments, the expected compound is isolated byflash chromatography on a silica gel column (eluent: CH₂Cl₂/ether 30%).11 is collected in the form of a colorless foam with a yield of 86%.R_(f) (AcOEt/petroleum ether, 7/3): 0.5. [α]_(D) ²⁰=+8.5° (c 1.29,CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.21 (s, 9H, C(CH₃)₃), 1.90 (s, 3H, NCOCH₃), 2.49(dd, J=5.9 and 12.9 Hz, 1H, □ Ha cys), 2.78 (dd, J=6.5and 12.9 Hz, 1H, βHb cys), 2.88-2.98 (m, 2H, NCH₂CH₂S), 3.28-3.40 (m, 2H, NCH₂CH₂S),4.06-4.19 (m, 1H, α H cys), 5.77 (d, J=7.6 Hz, 1H, NH cys), 6.27-6.41(m, 1H, NHCH₂), 7.16-7.35 and 7.40-7.48 (2m, 15H, aromatic H). MS:(FAB⁺/G-T) m/z 549 (M+H)⁺.

Analysis: C₃₁H₃₆N₂O₃S₂ (548) Calc. %: C 67.88 H 6.57 N 5.11 Found %:66.73 6.70 5.172.11. N-(N-Acetyl-L-cysteinyl)-S-pivaloylcysteamine (I-193)

This compound is obtained by S-detritylation of 11 (4.57 mmol). Theprotocol used is the same as that described in example 1 for thesynthesis of I-152. After the various treatments, a gum is collected,which gum is purified by flash chromatography on a silica gel column(eluent: CH₂Cl₂/MeOH 1.5%). I-193 is isolated in the form of a colorlesssemicrystalline gum with a yield of 60%.

R_(f) (CH₂Cl₂/MeOH, 9.5/0.5): 0.49. [α]_(D) ²⁰=−20.2° (c 0.94, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.24 (s, 9H, C(CH₃)₃), 1.61 (dd, J=7.6 and 10.3Hz, 1H, SH), 2.08 (s, 3H, NCOCH₃), 2.70 (ddd, J=6.4, 10.3 and 13.9 Hz,1H, β Ha cys), 2.97-3.09 (m, 2H, NCH₂CH₂S), 3.08 (ddd, J=4.1, 7.6 and13.9 Hz, 1H, β Hb cys), 3.40-3.52 (m, 2H, NCH₂CH₂S), 4.60 (ddd, J=4.1,6.4 and 7.8 Hz, 1H, α H cys), 6.49 (d, J=7.8 Hz, 1H, NH cys), 6.69-6.82(m, 1H, NHCH₂). MS: (FAB⁺/G-T) m/z 613 (2M+H)⁺, 307 (M+H)⁺.

Analysis: C₁₂H₂₂N₂O₃S₂ (306) Calc. %: C 47.06 H 7.19 N 9.15 Found %:47.37 7.23 9.222.12. N-(N,S-Bisacetyl-L-cysteinyl)-S-pivaloylcysteamine (I-194)

The S-acylation of I-193 (0.33 mmol) with acetic anhydride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum is purified by flash chromatography on a silica gelcolumn (eluent: CH₂Cl₂/ether 55%). I-194 is isolated in the form of agum (Yd=71%) which crystallizes from an AcOEt/petroleum ether mixture incolorless platelets. R_(f) (CH₂Cl₂/MeOH, 9.5/0.5): 0.58. M.p.=112-114°C. [α]_(D) ²⁰=−13.8° (c 0.94, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.24 (s, 9H, C(CH₃)₃), 2.03 (s, 3H, NCOCH₃), 2.38(s, 3H, SCOCH₃), 2.94-3.04 (m, 2H, NCH₂CH₂S), 3.18-3.36 (m, 2H, CH₂cys), 3.36-3.48 (m, 2H, NCH₂CH₂S), 4.48-4.60 (m, 1H, α H cys), 6.40 (d,J=7.5 Hz, 1H, NH cys), 6.71-6.83 (m, 1H, NHCH₂). MS: (FAB⁺/G-T) m/z 697(2M+H)⁺, 349 (M+H)⁺.

Analysis: C₁₄H₂₄N₂O₄S₂ (348) Calc. %: C 48.28 H 6.90 N 8.05 Found %:48.34 7.00 7.972.13. N-(N-Acetyl-S-isobutyryl-L-cysteinyl)-S-pivaloylcysteamine (I-195)

The S-acylation of I-193 (0.32 mmol) with isobutyryl chloride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum, after triturations from hexane, provides acolorless powder which is homogeneous by TLC(Yd=56%). R_(f)(CH₂Cl₂/ether, 3.5/6.5): 0.46. M.p.=101-102° C. [α]_(D) ²⁰=−5.7° (c1.05, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.20 (d, J=6.9 Hz, 6H, C(CH₃)₂), 1.25 (s, 9H,C(CH₃)₃), 2.01 (s, 3H, NCOCH₃), 2.79 (hept, J=6.9 Hz, 1H, CH(CH₃)₂),2.90-3.08 (m, 2H, NCH₂CH₂S), 3.25 (d, J=6.3 Hz, CH₂ cys), 3.35-3.47 (m,2H, NCH₂CH₂S), 4.46-4.58 (m, 1H, α H cys), 6.38 (d, J=7.1 Hz, 1H, NHcys), 6.71-6.81 (m, 1H, NHCH₂). MS: (FAB⁺/G-T) m/z 753 (2M+H)⁺, 377(M+H)⁺.

Analysis: C₁₆H₂₈N₂O₄S₂ (376) Calc. %: C 51.06 H 7.45 N 7.45 Found %:51.20 7.49 7.462.14. N-(N-Acetyl-S-pivaloyl-L-cysteinyl)-S-pivaloylcysteamine (I-196)

The S-acylation of I-193 (0.34 mmol) with pivaloyl chloride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum, after triturations in hexane, provides a colorlesspowder (Yd=66%). R_(f) (CH₂Cl₂/ether, 5/5): 0.36. Crystallizes from anAcOEt/petroleum ether mixture in the form of colorless microcrystals.M.p.=109-111° C. [α]_(D) ²⁰=−4.4° (c 0.91, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.24 (s, 18H, C(CH₃)₃), 2.00 (s, 3H, NCOCH₃),2.90-3.08 (m, 2H, NCH₂CH₂S), 3.24 (d, J=6.4 Hz, 2H, CH₂ cys), 3.36-3.47(m, 2H, NCH₂CH₂S), 4.44-4.57 (m, 1H, α H cys), 6.38 (d, J=7.4 Hz, 1H, NHcys), 6.68-6.88 (m, 1H, NHCH₂). MS: (FAB⁺/G-T) m/z 781 (2M+H)⁺, 391(M+H)⁺.

Analysis: C₁₇H₃₀N₂O₄S₂ (390) Calc. %: C 52.31 H 7.69 N 7.18 Found %:52.47 7.70 7.142.15. N-(N-Acetyl-S-benzoyl-L-cysteinyl)-S-pivaloylcysteamine (I-197)

The S-acylation of I-193 (0.34 mmol) with benzoyl chloride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum is purified by flash chromatography on a silica gelcolumn (eluent: CH₂Cl₂/ether 35%). I-197 is isolated in the form of agum which, after trituration in hexane, provides a colorless powder(Yd=66%). R_(f) (CH₂Cl₂/ether, 5/5): 0.33. M.p.=133-134° C. [α]_(D)²⁰=+5.10° (c 0.98, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.21 (s, 9H, C(CH₃)₃); 2.01 (s, 3H, NCOCH₃),2.90-3.08 (m, 2H, NCH₂CH₂S), 3.33-3.52 (m, 4H, NCH₂CH₂S, CH₂ cys),4.64-4.77 (m, 1H, α H cys), 6.76 (d, J=7.4 Hz, 1H, NH cys), 7.06-7.22(m, 1H, NHCH₂), 7.40-7.50, 7.55-7.63, 7.91-8.0 (3m, 5H, aromatic H). MS:(FAB⁺/G-T) m/z 821 (2M+H)⁺, 411 (M+H)⁺.

Analysis: C₁₉H₂₆N₂O₄S₂ (410) Calc. %: C 55.61 H 6.34 N 6.83 Found %:55.29 6.35 6.752.16. N-(N-Acetyl-S-trityl-L-cysteinyl)-S-benzoylcysteamine (12)

The coupling reaction of 7 (7.41 mmol) is carried out according toMethod B described in the first synthetic route (example 1):S-acetylcysteamine hydrochloride was replaced by S-benzoylcysteaminehydrochloride (T. Wieland and E. Bokelman, Ann. Chem., 1952, 576, 20-34.After the various treatments, the expected compound is isolated by flashchromatography on a silica gel column (eluent: CH₂Cl₂/ether 15%). 12 iscollected in the form of a colorless foam with a yield of 62%. R_(f)(AcOEt/petroleum ether, 7/3): 0.42. [α]_(D) ²⁰=+10.8° (c 1.11, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.86 (s, 3H, NCOCH₃), 2.47 (dd, J=5.7 and 13.0 Hz,1H, β Ha cys), 2.82 (dd, J=6.4 and 13.0 Hz, 1H, β Hb cys), 3.08-3.27 (m,2H, NCH₂CH₂S), 3.41-3.53 (m, 2H, NCH₂CH₂S), 4.08-4.21 (m, 1H, α H cys),5.67 (d, J=7.7 Hz, 1H, NH cys), 6.34-6.46 (m, 1H, NHCH₂), 7.17-7.32,7.37-7.45, 7.54-7.61, 7.89-7.96 (4m, 20H, aromatic H). MS: (FAB⁺/G-T)m/z 569 (M+H)⁺.

Analysis: C₃₃H₃₂N₂O₃S₂ (568) Calc. %: C 69.72 H 5.63 N 4.93 Found %:68.96 5.62 4.892.17. N-(N-Acetyl-L-cysteinyl)-S-benzoylcysteamine (I-198)

This compound is obtained by the S-detritylation of 12 (4.44 mmol). Theprotocol used is the same as that described in example 1 for thesynthesis of I-152. After the various treatments, a gum is collected,which gum is purified by flash chromatography on a silica gel column(eluent: AcOEt/petroleum ether 50%). I-198 is isolated in the form of awhite solid with a yield of 63%. R_(f) CH₂Cl₂/MeOH, 9.5/0.5): 0.38.M.p.=128-130° C. [α]_(D) ²⁰=−24.7° (c 1.01, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.59 (dd, J=7.6 and 10.2 Hz, 1H, SH), 2.04 (s, 3H,NCOCH₃), 2.71 (ddd, J=6.5, 10.2 and 13.8 Hz, 1H, β Ha cys), 3.06 (ddd,J=4.3, 7.6 and 13.8 Hz, 1H, β Hb cys), 3.20-3.31 (m, 2H, NCH₂CH₂S),3.52-3.64 (m, 2H, NCH₂CH₂S), 4.61 (ddd, J=4.3, 6.5 and 7.4 Hz, 1H, α Hcys), 6.51 (d, J=7.4 Hz, 1H, NH cys), 6.83-7.00 (m, 1H, NHCH₂),7.43-7.52, 7.56-7.65, 7.92-8.00 (3m, 5H, aromatic H). MS: (FAB⁺/G-T) m/z653 (2M+H)⁺, 327 (M+H)⁺.

Analysis: C₁₄H₁₈N₂O₃S₂ (326) Calc. %: C 51.53 H 5.52 N 8.59 Found %:51.49 5.55 8.602.18. N-(N,S-Bisacetyl-L-cysteinyl)-S-benzoylcysteamine (I-199)

The S-acylation of I-198 (0.34 mmol) with acetic anhydride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum is purified by flash chromatography on a silica gelcolumn (eluent: CH₂Cl₂/MeOH 1.5%). I-199 is isolated in the form of agum (Yd=75%) which crystallizes from AcOEt in colorless needles. R_(f)(CH₂Cl₂/MeOH, 9.5/0.5): 0.44. M.p.=166-168° C. [α]_(D) ²⁰=−14.3° (c0.98, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.99 (s, 3H, NCOCH₃), 2.32 (s, 3H, SCOCH₃),3.18-3.31 (m, 4H, NCH₂CH₂S, CH₂ cys), 3.48-3.61 (m, 2H, NCH₂CH₂S),4.49-4.62 (m, 1H, α H cys), 6.38 (d, J=7.6 Hz, 1H, NH cys), 6.79-6.92(m, 1H, NHCH₂), 7.42-7.51, 7.55-7.64, 7.93-8.01 (3m, 5H, aromatic H).MS: (FAB⁺/G-T) m/z 737 (2M+H)⁺, 369 (M+H)⁺.

Analysis: C₁₆H₂₀N₂O₄S₂ (368) Calc. %: C 52.17 H 5.43 N 7.61 Found %:52.33 5.45 7.682.19. N-(N-Acetyl-S-isobutyryl-L-cysteinyl)-S-benzoylcysteamine (I-200)

The S-acylation of I-198 (0.30 mmol) with isobutyryl chloride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum is purified by flash chromatography on a silica gelcolumn (eluent: CH₂Cl₂/ether 40%). I-200 is isolated in the form of agum (Yd=79%) which crystallizes from an AcOEt/petroleum ether mixture incolorless platelets. R_(f) (CH₂Cl₂/ether, 3/7): 0.2. M.p.=135-136° C.[α]_(D) ²⁰=−6.7° (c 1.2, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.17 (d, J=6.9 Hz, 6H, C(CH₃)₂), 1.97 (s, 3H,NCOCH₃), 2.75 (hept, J=6.9 Hz, 1H, CH(CH₃)₂), 3.19-3.30 (m, 4H,NCH₂CH₂S, CH₂ cys), 3.46-3.62 (m, 2H, NCH₂CH₂S), 4.49-4.61 (m, 1H, α Hcys), 6.41 (d, J=7.4 Hz, 1H, NH cys), 6.85-6.96 (m, 1H, NHCH₂),7.41-7.52, 7.55-7.64, 7.90-8.02 (3m, 5H, aromatic H). MS: (FAB⁺/G-T) m/z793 (2M+H)⁺, 397 (M+H)⁺.

Analysis: C₁₈H₂₄N₂O₄S₂ (396) Calc. %: C 54.54 H 6.06 N 7.07 Found %:54.77 6.04 7.072.20. N-(N-Acetyl-S-pivaloyl-L-cysteinyl)-S-benzoylcysteamine (I-201)

The S-acylation of I-198 (0.30 mmol) with pivaloyl chloride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum is purified by flash chromatography on a silica gelcolumn (eluent: CH₂Cl₂/ether 45%). I-201 is isolated in the form of agum (Yd=83%) which crystallizes from an AcOEt/petroleum ether mixture incolorless platelets. R_(f) (CH₂Cl₂/ether, 3/7): 0.3. M.p.=101-103° C.[α]_(D) ²⁰=−3.8° (c 1.05, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.22 (s, 9H, C(CH₃)₃), 1.96 (s, 3H, NCOCH₃),3.19-3.30 (m, 4H, NCH₂CH₂S, CH₂ cys), 3.46-3.62 (m, 2H, NCH₂CH₂S),4.47-4.58 (m, 1H, α H cys), 6.38 (d, J=7.2 Hz, 1H, NH cys), 6.81-6.92(m, 1H, NHCH₂), 7.42-7.52, 7.55-7.65, 7.90-8.02 (3m, 5H, aromatic H).MS: (FAB⁺/G-T) m/z 821 (2M+H)⁺, 411 (M+H)⁺.

Analysis: C₁₉H₂₆N₂O₄S₂ (410) Calc. %: C 55.61 H 6.34 N 6.83 Found %:55.73 6.29 6.852.21. N-(N-Acetyl-S-benzoyl-L-cysteinyl)-S-benzoylcysteamine (I-202)

The S-acylation of I-198 (0.30 mmol) with benzoyl chloride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum is purified by flash chromatography on a silica gelcolumn (eluent: CH₂Cl₂/MeOH 1%). I-202 is isolated in the form of a gum(Yd=72%) which crystallizes from an AcOEt in colorless platelets. R_(f)(CH₂Cl₂/MeOH, 9.5/0.5): 0.66. M.p.=188-190° C. [α]_(D) ²⁰=+4.1° (c 0.98,CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.99 (s, 3H, NCOCH₃), 3.19-3.28 (m, 2H, NCH₂CH₂S),3.49 (d, J=6.1 Hz, 2H, CH₂ cys), 3.52-3.61 (m, 2H, NCH₂CH₂S), 4.63-4.71(m, 1H, α H cys), 6.56 (d, J=7.2 Hz, 1H, NH cys), 6.92-7.08 (m, 1H,NHCH₂), 7.38-7.48, 7.52-7.62, 7.88-7.97 (3m, 10H, aromatic H). MS:(FAB⁺/G-T) m/z 861 (2M+H)⁺, 431 (M+H)⁺.

Analysis: C₂₁H₂₂N₂O₄S₂ (430) Calc. %: C 58.60 H 5.12 N 6.51 Found %:58.37 5.10 6.512.22. N-Isobutyryl-S-trityl-L-cysteine (13)

This compound was synthesized by adapting, to N-isobutyryl-L-cysteine,the tritylation method described by K.-Y. Zee-Cheng and C. C. Cheng, J.Med. Chem., 1970, 13, 414-418. A suspension comprising 4.1 g (21.5 mmol)of N-isobutyryl-L-cysteine [(prepared according to H. Brückner et al.,J. Chromatogr., 1989, 476, 73-82), ([α]_(D) ²⁰=+84° (c 1, CHCl₃))], 5.6g (21.5 mmol) of triphenylmethanol and 16 ml of glacial acetic acid isstirred at ambient temperature. 4.1 ml (32.2 mmol) of BF₃ etherate areadded dropwise to the mixture while maintaining the temperature at20-25° C. After stirring for 3 h, the brown solution obtained issubsequently cooled to 0° C. and then 70 ml of an aqueous sodium acetatesolution and 140 ml of water are added. At the end of the additions, thereaction mixture sets solid in the form of a gel. This mixture is leftovernight at 0° C. and then 150 ml of ice-cold water and 120 ml of etherare subsequently added with vigorous stirring. The ethereal phase isseparated by settling and the aqueous waste liquors are washed with 4×80ml of ether. The organic phases are combined, washed with 4×60 ml ofice-cold water, dried over sodium sulfate, filtered and evaporated todryness under vacuum. The crude product obtained is purified by washingsuccessively with hexane (5×50 ml) to provide 13 in the form of a gumwhich is homogeneous by TLC with a yield of 81%. R_(f)(CH₂Cl₂/MeOH/AcOH, 9.4/0.6/0.07): 0.53. [α]_(D) ²⁰=+21.4° (c 1.12,CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.12, 1.14 (2d, J=2×6.9 Hz, 2×3H, C(CH₃) ₂),2.24-2.42 (m, 1H, CH(CH₃) ₂, 2.64-2.28 (m, 2H, CH₂), 4.31-4.43 (m, 1H, αH), 5.67-6.15 (broad m, 1H, CO₂H), 5.90 (d, J=7.1 Hz, 1H, NH partiallyconcealing the m at 5.67-6.15), 7.18-7.33, 7.37-7.46 (2m, 15H, aromaticH). MS: (FAB⁺/G-T) m/z 867 (2M+H)⁺, 431 (M+H)⁺; (FAB⁺/G-T) m/z 865(2M−H)⁻, 432 (M−H)⁺.

Analysis: C₂₆H₂₇NO₃S₂ (433) Calc. %: C 72.06 H 6.24 N 3.23 Found %:72.24 6.22 3.282.23. N-(N-Isobutyryl-S-trityl-L-cysteinyl)-S-acetylcysteamine (14)

The coupling reaction of 13 (3.93 mmol) with S-acetylcysteaminehydrochloride is carried out according to method B described in thefirst synthetic route (example 1). After the various treatments, theexpected compound is isolated by flash chromatography on a silica gelcolumn (eluent: AcOEt/petroleum ether 60%). 14 is collected in the formof a colorless foam with a yield of 67%. R_(f) (AcOEt/petroleum ether,6/4): 0.5. [α]_(D) ²⁰=+9.3° (c 0.97, CHCl₃).

¹H NMR CDCl₃) δ ppm: 1.10 (d, J=6.9 Hz, 6H, C(CH₃)₂), 2.18-2.36 (m, 1H,CH(CH₃)₂), 2.29 (s, 3H, SCOCH₃ partially concealing the m at 2.18-2.36),2.50 (dd, J=5.6 and 12.8 Hz, 1H, β Ha cys), 2.72 (dd, J=6.7 and 12.8 Hz,1H, β Hb cys), 2.88-3.01 (m, 2H, NCH₂CH₂S), 3.29-3.41 (m, 2H, NCH₂CH₂S),4.06-4.19 (m, 1H, α H cys), 5.82 (d, J=7.4 Hz, 1H, NH cys), 6.42 (t,J=5.5 Hz, 1H, NHCH₂), 7.17-7.35, 7.39-7.48 (2m, 15H, aromatic H). MS:(FAB⁺/G-T) m/z 535 (M+H)⁺.

Analysis: C₃₀H₃₄N₂O₃S (534) Calc. %: C 67.42 H 6.37 N 5.24 Found %:67.05 6.72 5.302.24. N-(N-Isobutyryl-L-cysteinyl)-S-acetylcysteamine (I-203)

This compound is obtained by the S-detritylation of 14 (2.56 mmol). Theprotocol used is the same as that described in example 1 for thesynthesis of I-152. After the various treatments, a gum is collected,which gum is purified by flash chromatography on a silica gel column(eluent: CH₂Cl₂/ether 30%). I-203 is isolated in the form of white solidwith a yield of 70%. R_(f) CH₂Cl₂/ether, 5/5): 0.44. M.p.=117-120° C.[α]_(D) ²⁰=−36.5° (c 1.04, CHCl₃).

¹H NMR CDCl₃) δ ppm: 1.19, 1.20 (2d, J=2×6.9 Hz, 2×3H, C(CH₃)₂), 1.62(dd, J=7.5 and 10.3 Hz, 1H, SH), 2.37 (s, 3H, SCOCH₃), 2.47 (hept, J=6.9Hz, 1H, CH(CH₃)₂), 2.72 (ddd, J=6.5, 10.3 and 13.9 Hz, 1H, β Ha cys),3.00-3.08 (m, 2H, NCH₂CH₂S), 3.06 (ddd, J=4.2, 7.5 and 13.9 Hz, 1H, β Hbcys), 3.41-3.53 (m, 2H, NCH₂CH₂S), 4.61 (ddd, J=4.2, 6.5 and 7.4 Hz, 1H,α H cys), 6.46 (d, J=7.4 Hz, 1H, NH cys), 6.72-6.85 (m, 1H, NHCH₂). MS:(FAB⁺/G-T) m/z 293 (M+H)⁺.

Analysis: C₁₁H₂₀N₂O₃S₂ (292) Calc. %: C 45.20 H 6.84 N 9.59 Found %:45.22 7.11 9.692.25. N-(N-Isobutyryl-S-acetyl-L-cysteinyl)-S-acetylcysteamine (I-204)

The S-acylation of I-203 (0.34 mmol) with acetic anhydride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum is purified by flash chromatography on a silica gelcolumn (eluent: CH₂Cl₂/ether 50%). I-204 is isolated in thesemicrystalline form (Yd=75%), which product, after trituration inhexane provides a colorless powder. R_(f) (CH₂Cl₂/ether, 2/8): 0.43.M.p.=125-127° C. [α]_(D) ²⁰=−22.9° (c 1.05, CHCl₃).

¹H NMR CDCl₃) δ ppm: 1.16 (d, J=6.9 Hz, 6H, C(CH₃)₂), 2.34-2.47 (m, 1H,CH(CH₃)₂), 2.36, 2.38 (2s, 2×3H, 2×SCOCH₃ partially concealing the m at2.34-2.47), 2.97-3.06 (m, 2H, NCH₂CH₂S), 3.26-3.32 (m, 2H, CH₂ cys),3.38-3.48 (m, 2H, NCH₂CH₂S), 4.47-4.58 (m, 1H, α H cys), 6.39 (d, J=7.3Hz, 1H, NH cys), 6.80-6.91 (m, 1H, NHCH₂). MS: (FAB⁺/G-T) m/z 669(2M+H)⁺, 335 (M+H)⁺.

Analysis: C₁₃H₂₂N₂O₄S₂ (334) Calc. %: C 46.71 H 6.59 N 8.33 Found %:46.76 6.90 8.352.26. N-(N,S-Bisisobutyryl-L-cysteinyl)-S-acetylcysteamine (I-205)

The S-acylation of I-203 (0.32 mmol) with isobutyryl chloride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, an oil iscollected, which oil is purified by flash chromatography on a silica gelcolumn (eluent: AcOEt/petroleum ether 70%). I-205 is isolated in theform of a colorless gum (Yd=56%). R_(f) (AcOEt/petroleum ether, 4/6):0.20. [α]_(D) ²⁰=−17.3° (c 1.1, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.16 (d, J=6.9 Hz, 6H, C(CH₃)₂ of N-i-but.), 1.20,1.21 (2d, J=2×6.9 Hz, 2×3H, C(CH₃)₂ of S-i-but), 2.33-2.46 (m, 1H,CH(CH₃)₂ of N-i-but), 2.36 (s, 3H, SCOCH₃ partially concealing the m at2.33-2.46); 2.79 (app. hept, J=6.9 Hz, 1H, CH(CH₃)₂ of S-i-but),2.97-3.07 (m, 2H, NCH₂CH₂S), 3.25 (dd, J=5.0 and 13.8 Hz, 1H, β Ha cys),3.32 (dd, J=7.5 and 13.8 H, 1H, β Hb cys), 3.38-3.48 (m, 2H, NCH₂CH₂S),4.45-4.57 (m, 1H, α H cys), 6.44 (d, J=7.1 Hz, 1H, NH cys), 6.86-6.97(m, 1H, NHCH₂). MS: (FAB⁺/G-T) m/z 725 (2M+H)⁺, 363 (M+H)⁺.

Analysis: C₁₅H₂₆N₂O₄S₂ (362) Calc. %: C 49.72 H 7.18 N 7.73 Found %:49.87 7.36 7.802.27. N-(N-Isobutyryl-S-pivaloyl-L-cysteinyl)-S-acetylcysteamine (I-206)

The S-acylation of I-203 (0.31 mmol) with pivaloyl chloride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum is purified by flash chromatography on a silica gelcolumn (eluent: CH₂Cl₂/ether 50%). I-206 is isolated in thesemicrystalline form (Yd=60%), which form, after trituration in hexane,provides a colorless powder. R_(f) (AcOEt/petroleum ether, 4/6): 0.3.M.p.=101-103° C. [α]_(D) ²⁰=−19.3° (c 1.09, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.15 (d, J=6.9 Hz, 6H, C(CH₃)₂), 1.24 (s, 9H,C(CH₃)₃), 2.30-2.45 (m, 1H, CH(CH₃)₂), 2.36 (s, 3H, SCOCH₃ partiallyconcealing the m at 2.30-2.45), 2.96-3.07 (m, 2H, NCH₂CH₂S), 3.23 (dd,J=5.3 and 13.8 Hz, 1H, β Ha cys), 3.30 (dd, J=6.9 and 13.8 Hz, 1H, β Hbcys), 3.37-3.49 (m, 2H, NCH₂CH₂S), 4.44-4.57 (m, 1H, α H cys), 6.44 (d,J=7.1 Hz, 1H, NH cys), 6.85-6.98 (m, 1H, NHCH₂). MS: (FAB⁺/G-T) m/z 753(2M+H)⁺, 377 (M+H)⁺.

Analysis: C₁₆H₂₈N₂O₄S₂ (376) Calc. %: C 51.06 H 7.45 N 7.45 Found %:50.98 7.72 7.542.28. N-(N-Isobutyryl-S-benzoyl-L-cysteinyl)-S-acetylcysteamine (I-207)

The S-acylation of I-203 (0.31 mmol) with benzoyl chloride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum is purified by flash chromatography on a silica gelcolumn (eluent: AcOEt/petroleum ether 75%). I-207 is isolated in theform of a colorless solid (Yd=55%) which crystallizes from ether incolorless microcrystals. R_(f) (CH₂Cl₂/ether, 7.5/2.5): 0.45.M.p.=135-137° C. [α]_(D) ²⁰=+3.5° (c 1.16, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.12, 1.14 (2d, J=2×6.9 Hz, 2×3H, C(CH₃)₂),2.30-2.47 (m, 1H, CH(CH₃)₂), 2.33 (s, 3H, SCOCH₃ partially concealingthe m at 2.30-2.47), 2.98-3.07 (m, 2H, NCH₂CH₂S), 3.40-3.58 (m, 4H, CH₂cys, NCH₂CH₂S), 4.59-4.70 (m, 1H, α H cys), 6.59 (d, J=7.1 Hz, 1H, NHcys), 6.93-7.04 (m, 1H, NHCH₂), 7.41-7.53, 7.57-7.61, 7.92-8.01 (3m, 5H,aromatic H). MS: (FAB⁺/G-T) m/z 793 (2M+H)⁺, 397 (M+H)⁺.

Analysis: C₁₈H₂₄N₂O₄S₂ (396) Calc. %: C 54.54 H 6.06 N 7.07 Found %:54.51 6.20 7.072.29. N-(N,-Isobutyryl-S-trityl-L-cysteinyl)-S-isobutyrylcysteamine (15)

The coupling reaction of 13 (3.93 mmol) with S-isobutyrylcysteaminehydrochloride is carried out according to method B described in thefirst synthetic route (example 1). After the various treatments, theexpected compound is isolated by flash chromatography on a silica gelcolumn (eluent: AcOEt/petroleum ether 65%). 15 is collected in the formof a colorless foam with a yield of 75%. R_(f) (AcOEt/petroleum ether,5/5): 0.6. [α]_(D) ²⁰=+7.9° (c 1.27, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.107, 1.110, 1.159, 1.162 (4d, J=4×6.9 Hz, 4×3H,2×C(CH₃)₂, 2.17-2.32 (m, 1H, CH(CH₃)₂ of N-i-but), 2.51 (dd, J=5.7 and12.8 Hz, 1H, β Ha cys), 2.63-2.79 (m, 1H, CH(CH₃)₂ of S-i-but), 2.72(dd, J=6.8 and 12.8 Hz, 1H, β Hb cys partially concealing the m at2.63-2.79), 2.88-2.98 (m, 2H, NCH₂CH₂S), 3.29-3.40 (m, 2H, NCH₂CH₂S),4.07-4.19 (m, 1H, α H cys), 5.81 (d, J=7.3 Hz, 1H, NH cys), 6.32-6.43(m, 1H, NHCH₂), 7.18-7.35, 7.39-7.47 (2m, 15H, aromatic H). MS:(FAB⁺/G-T) m/z 563 (M+H)⁺.

Analysis: C₃₂H₃₈N₂O₃S₂ (562) Calc. %: C 68.33 H 6.76 N 4.98 Found %:68.27 6.71 4.882.30. N-(N-Isobutyryl-L-cysteinyl)-S-isobutyrylcysteamine (I-208)

This compound is obtained by the S-detritylation of 15 (2.75 mmol). Theprotocol used is the same as that described in example 1 for thesynthesis of I-152. After the various treatments, a gum is collected,which gum is purified by flash chromatography on a silica gel column(eluent: CH₂Cl₂/ether 25%). I-208 is isolated in the form of a gumwhich, after trituration in hexane, provides a colorless powder(Yd=69%). R_(f) (CH₂Cl₂/ether, 5/5): 0.39. M.p.=125-127° C. [α]_(D)²⁰=−25.7° (c 1.05, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.19, 1.20 (2d; J=2×6.9 Hz, 2×6H, 2×C(CH₃)₂), 1.62(dd, J=7.5 and 10.3 Hz, 1H, SH), 2.41-2.54 (m, 1H, CH(CH₃)₂ of N-i-but),2.70-2.84 (m, 1H, CH(CH₃)₂ of S-i-but), 2.72 (ddd, J=6.5, 10.3 and 13.7Hz, 1H, β Ha cys partially concealing the m at 2.70-2.84), 2.98-3.14 (m,3H, β Hb cys, NCH₂CH₂S), 3.42-3.53 (m, 2H, NCH₂CH₂S), 4.54-4.66 (m, 1H,α H cys), 5.81 (d, J=7.4 Hz, 1H, NH cys), 6.74-6.85 (m, 1H, NHCH₂). MS:(FAB⁺/G-T) m/z 641 (2M+H)⁺, 321 (M+H)⁺.

Analysis: C₁₃H₂₄N₂O₃S₂ (320) Calc. %: C 48.75 H 7.50 N 8.75 Found %:48.45 7.82 8.752.31. N-(N-Isobutyryl-S-acetyl-L-cysteinyl)-S-isobutyrylcysteamine(I-209)

The S-acylation of I-208 (0.28 mmol) with acetic anhydride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum, after triturations in hexane, provides a colorlesspowder which is homogeneous by TLC with a yield of 80%. R_(f)(CH₂Cl₂/ether, 5/5): 0.55. M.p.=100-102° C. [α]_(D) ²⁰=−22.9° (c 0.92,CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.15, 1.20 (2d, J=2×6.9 Hz, 2×6H, 2×C(CH₃)₂),2.34-2.47 (m, 1H, CH(CH₃)₂ of N-i-but), 2.37 (s, 3H, SCOCH₃ partiallyconcealing the m at 2.34-2.47), 2.69-2.82 (m, 1H, CH(CH₃)₂ of S-i-but),2.95-3.05 (m, 2H, NCH₂CH₂S), 3.26-3.32 (m, 2H, CH₂ cys), 3.37-3.47 (m,2H, NCH₂CH₂S), 4.47-4.59 (m, 1H, α H cys), 6.39 (d, J=7.1 Hz, 1H, NHcys), 6.79-6.90 (m, 1H, NHCH₂). MS: (FAB⁺/G-T) m/z 725 (2M+H)⁺, 363(M+H)⁺.

Analysis: C₁₅H₂₆N₂O₄S₂ (362) Calc. %: C 49.72 H 7.18 N 7.73 Found %:49.47 7.41 7.702.31. N-(N,S-Bisisobutyryl-L-cysteinyl)-S-isobutyrylcysteamine (I-210)

The S-acylation of I-208 (0.28 mmol) with isobutyryl chloride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum is purified by flash chromatography on a silica gelcolumn (eluent: CH₂Cl₂/ether 25%). I-210 is isolated in the form of agum which, after trituration in hexane, provides a colorless powder(Yd=63%). R_(f) (CH₂Cl₂/ether, 5/5): 0.68. M.p.=94-96° C. [α]_(D)²⁰=−11° (c 0.91, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.154, 1.158, 1.199, 1.202 (4d, J=4×6.9 Hz, 2×3H,2×6H, 3×C(CH₃)₂), 2.32-2.46 (m, 1H, CH(CH₃)₂ of N-i-but), 2.69-2.86 (m,2H, 2×CH(CH₃)₂ of 2×S-i-but), 2.93-3.07 (in, 2H, NCH₂CH₂S), 3.25 (dd,J=5.0 and 14.5 Hz, 1H, β Ha cys), 3.32 (dd, J=7.7 and 14.5 Hz, 1H, β Hbcys), 3.38-3.47 (m, 2H, NCH₂CH₂S), 4.45-4.56 (m, 1H, α H cys), 6.42 (d,J=7.4 Hz, 1H, NH cys), 6.82-6.92 (m, 1H, NHCH₂). MS: (FAB⁺/G-T) m/z 781(2M+H)⁺, 391 (M+H)⁺.

Analysis: C₁₇H₃₀N₂O₄S₂ (390) Calc. %: C 52.31 H 7.69 N 7.18 Found %:52.02 8.02 7.212.32. N-(N-Isobutyryl-S-benzoyl-L-cysteinyl)-S-isobutyrylcysteamine(I-211)

The S-acylation of I-208 (0.29 mmol) with benzoyl chloride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum is purified by flash chromatography on a silica gelcolumn (eluent: CH₂Cl₂/ether 35%). I-210 is isolated in the form of agum which, after trituration in petroleum ether, provides a colorlesspowder (Yd=86%). R_(f) (CH₂Cl₂/ether, 6/4): 0.52. M.p.=155-157° C.[α]_(D) ²⁰=+7.0° (c 1, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.12, 1.15, 1.18 (3d, J=3×6.9 Hz, 2×3H, 1×6H,2×C(CH₃)₂), 2.36-2.46 (m, 1H, CH(CH₃)₂ of N-i-but), 2.68-2.80 (m, 1H,CH(CH₃)₂ of S-i-but), 2.95-3.05 (m, 2H, NCH₂CH₂S), 3.40-3.62 (m, 4H, CH₂cys, NCH₂CH₂S), 4.56-4.69 (m, 1H, α H cys), 6.54 (d, J=7.0 Hz, 1H, NHcys), 6.84-6.95 (m, 1H, NHCH₂), 7.42-7.51, 7.57-7.65, 7.94-8.01 (m, 5H,aromatic H). MS: (FAB⁺/G-T) m/z 894 (2M+H)⁺, 425 (M+H)⁺.

Analysis: C₂₀H₂₈N₂O₄S₂ (424) Calc. %: C 56.60 H 6.60 N 6.60 Found %:56.42 6.79 6.632.33. N-(N-Isobutyryl-S-trityl-L-cysteinyl)-S-pivaloylcysteamine (16)

The coupling reaction of 13 (3.93 mmol) of S-pivaloylcysteaminehydrochloride is carried out according to method B described in thefirst synthetic route (example 1). After the various treatments, theexpected compound is isolated by flash chromatography on a silica gelcolumn (eluent: AcOEt/petroleum ether 70%). 16 is collected in the formof a foam which, after trituration in hexane, provides a colorlesspowder (Yd=88%). R_(f) (CH₂Cl₂/ether, 6/4): 0.82. M.p.=85-88° C. [α]_(D)²⁰=+5.9° (c 1.02, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.11 (d, J=6.9 Hz, 6H, C(CH₃)₂), 1.21 (s, 9H,C(CH₃)₃), 2.20-2.38 (m, 1H, CH(CH₃)₂), 2.51 (dd, J=5.6 and 12.9 Hz, 1H,β Ha cys), 2.72 (dd, J=6.7 and 12.9 Hz, 1H, β Hb cys), 2.84-2.96 (m, 2H,NCH₂CH₂S), 3.27-3.39 (m, 2H, NCH₂CH₂S), 4.03-4.17 (m, 1H, α H cys), 5.78(d, J=7.4 Hz, 1H, NH cys), 6.22-6.34 (m, 1H, NHCH₂), 7.18-7.35,7.38-7.48 (2m, 15H, aromatic H). MS: (FAB⁺/G-T) m/z 577 (M+H)⁺.

Analysis: C₃₃H₄₀N₂O₃S₂ (576) Calc. %: C 68.75 H 6.94 N 4.86 Found %:68.49 6.98 4.932.34. N-(N-Isobutyryl-L-cysteiyl-S-pivaloylcysteamine (I-214)

This compound is obtained by the S-detritylation of 16 (2.78 mmol) Theprotocol used is the same as that described in example 1 for thesynthesis of I-152. After the various treatments, a gum is collected,which gum is purified by flash chromatography on a silica gel column(eluent: CH₂Cl₂/ether 22%). I-214 is isolated in the form of a gumwhich, after trituration in hexane, provides a colorless powder(Yd=70%). R_(f) (CH₂Cl₂/ether, 5/5): 0.46. M.p.=120-122° C. [α]_(D)²⁰=−25° (c 1.04, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.194, 1.198 (2d, J=2×6.9 Hz, 2×3H, C(CH₃)₂), 1.23(s, 9H, C(CH₃)₃), 1.61 (dd, J=7.6 and 10.2 Hz, 1H, SH), 2.47 (app. hept,J=6.9 Hz, 1H, CH(CH₃)₂), 2.72 (ddd, J=6.5, 10.2 and 13.8 Hz, 1H, β Hacys), 2.95-3.13 (m, 3H, β Hb cys, NCH₂CH₂S), 3.40-3.52 (m, 2H,NCH₂CH₂S), 4.54-4.66 (m, 1H, α H cys), 6.49 (d, J=7.5 Hz, 1H, NH cys),6.73-6.88 (m, 1H, NHCH₂). MS: (FAB⁺/G-T) m/z 669 (2M+H)⁺, 335 (M+H)⁺.

Analysis: C₁₄H₂₆N₂O₃S₂ (334) Calc. %: C 50.30 H 7.78 N 8.38 Found %:50.19 7.92 8.352.35. N-(N-Isobutyryl-S-acetyl-L-cysteinyl)-S-pivaloylcysteamine (I-215)

The S-acylation of I-214 (0.27 mmol) with acetic anhydride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum, after triturations in hexane, provides a colorlesspowder which is homogeneous by TLC with a yield of 80%. R_(f)(CH₂Cl₂/ether, 6/4): 0.45. M.p.=112-114° C. [α]_(D) ²⁰=−18.8° (c 0.9,CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.16 (d, J=6.9 Hz, 6H, C(CH₃)₂), 1.24 (s, 9H,C(CH₃)₃), 2.31-2.49 (m, 1H, CH(CH₃)₂), 2.37 (s, 3H, SCOCH₃ partiallyconcealing the m at 2.31-2.49), 2.88-3.08 (m, 2H, NCH₂CH₂S), 3.25-3.34(m, 2H, CH₂ cys), 3.35-3.47 (m, 2H, NCH₂CH₂S), 4.48-4.60 (m, 1H, α Hcys), 6.38 (d, J=7.3 Hz, 1H, NH cys), 6.74-6.89 (m, 1H, NHCH₂). MS:(FAB⁺/G-T) m/z 377 (M+H)⁺.

Analysis: C₁₅H₂₈N₂O₄S₂ (376) Calc. %: C 51.06 H 7.45 N 7.45 Found %:50.97 7.81 7.472.36. N-(N,S-Bisisobutyryl-L-cysteinyl)-S-pivaloylcysteamine (I-216)

The S-acylation of I-214 (0.26 mmol) with isobutyryl chloride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum is purified by flash chromatography on a silica gelcolumn (eluent: CH₂Cl₂/ether 15%). I-216 is isolated in the form of agum which, after trituration in hexane, provides a colorless powder(Yd=78%). R_(f) (CH₂Cl₂/ether, 6/4): 0.61. M.p.=105-107° C. [α]_(D)²⁰=−12.2° (c 1.07,CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.154, 1.157, 1.196, 1.201 (4d, J=4×6.9 Hz, 4×3H,2×C(CH₃)₂), 1.24 (s, 9H, C(CH₃)₃), 2.39 (app. hept, J=6.9 Hz, 1H,CH(CH₃)₂ of N-i-but), 2.79 (app. hept, J=6.9 Hz, 1H, CH(CH₃)₂ ofS-i-but), 2.87-3.08 (m, 2H, NCH₂CH₂S), 3.25 (dd, J=5.2 and 14.5 Hz, 1H,β Ha cys), 3.32 (dd, J=7.5 and 14.5 Hz, 1H, β Hb cys), 3.37-3.47 (m, 2H,NCH₂CH₂S), 4.46-4.59 (m, 1H, α H cys), 6.43 (d, J=7.0 Hz, 1H, NH cys),6.81-6.93 (m, 1H, NHCH₂). MS: (FAB⁺/G-T) m/z 809 (2M+H)⁺, 405 (M+H)⁺.

Analysis: C₁₈H₃₂N₂O₄S₂ (404) Calc. %: C 53.47 H 7.92 N 6.93 Found %:53.33 8.09 6.952.37. N-(N-Isobutyryl-S-pivaloyl-L-cysteinyl)-S-pivaloylcysteamine(I-217)

The S-acylation of I-214 (0.26 mmol) with pivaloyl chloride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum, after triturations in hexane, provides a colorlesspowder which is homogeneous by TLC with a yield of 55%. R_(f)(CH₂Cl₂/ether, 6/4): 0.63. M.p.=106-108° C. [α]_(D) ²⁰=−10.2° (c 1.18,CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.151, 1.157 (2d, J=2×6.9 Hz, 2×3H, C(CH₃)₂),1.240, 1.244 (2s, 2×9H, 2×C(CH₃)₃), 2.38 (app, hept, J=6.9 Hz, 1H,CH(CH₃)₂), 2.86-3.07 (m, 2H, NCH₂CH₂S), 3.24 (dd, J=5.0 and 14.2 Hz, 1H,β Ha cys), 3.30 (dd, J=6.5 and 14.2 Hz, 1H, β Hb cys), 3.33-3.48 (m, 2H,NCH₂CH₂S), 4.42-4.54 (m, 1H, α H cys), 6.39 (d, J=7.0 Hz, 1H, NH cys),6.74-6.86 (m, 1H, NHCH₂). MS: (FAB⁺/G-T) m/z 837 (2M+H)⁺, 419 (M+H)⁺.

Analysis: C₁₉H₃₄N₂O₄S₂ (418) Calc. %: C 54.81 H 8.17 N 6.73 Found %:54.50 8.27 6.742.38 (N-N-Isobutyryl-S-benzoyl-L-cysteinyl)-S-pivaloylcysteamine (I-218)

The S-acylation of I-214 (0.26 mmol) with benzoyl chloride is carriedout according to the general method described in example 2. The reactionmixture, is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum is purified by flash chromatography on a silica gelcolumn (eluent: CH₂Cl₂/ether 25%). I-218 is isolated in the form of agum which, after trituration in hexane, provides a colorless powder(Yd=73%). R_(f) (CH₂Cl₂/ether, 5/5): 0.57. M.p.=123-124° C. [α]_(D)²⁰=+8.7° (c 0.92, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.12, 1.14 (2d, J=2×6.9 Hz, 2×3H, C(CH₃)₂), 1.22(s, 9H, C(CH₃)₃), 2.40 (app. hept, J=6.9 Hz, 1H, CH(CH₃)₂), 2.89-3.09(m, 2H, NCH₂CH₂S), 3.37-3.61 (m, 4H, NCH₂CH₂S, CH₂ cys), 4.57-4.70 (m,1H, α H cys), 6.55 (d, J=7.3 Hz, 1H, NH cys), 6.84-6.94 (m, 1H, NHCH₂),7.41-7.52, 7.55-7.66, 7.92-8.01 (3m, 5H, aromatic H). MS: (FAB⁺/G-T) m/z877 (2M+H)⁺, 439 (M+H)⁺.

Analysis: C₂₁H₃₀N₂O₄S₂ (438) Calc. %: C 57.53 H 6.85 N 6.39 Found %:57.53 6.89 6.372.39. N-(N-Isobutyryl-S-trityl-L-cysteinyl)-S-benzoylcysteamine (17)

The coupling reaction of 13 (3.93 mmol) with S-benzoylcysteaminehydrochloride is carried out according to method B described in thefirst synthetic route (example 1). After the various treatments, theexpected compound is isolated by flash chromatography on a silica gelcolumn (eluent: AcOEt/petroleum ether 70%). 17 is collected in the formof a foam which, after trituration in hexane, provides a colorless foam(Yd=77%). R_(f) (CH₂Cl₂/ether, 6/4):0.76. [α]_(D) ²⁰=+7.8° (c 1.03,CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.09 (d, J=6.9 Hz, 6H, C(CH₃)₂), 2.18-2.31 (m, 1H,CH(CH₃)₂), 2.51 (dd, J=5.6 and 12.9 Hz, 1H, β Ha cys), 2.74 (dd, J=6.7and 12.9 Hz, 1H, β Hb cys), 3.07-3.25 (m, 2H, NCH₂CH₂S), 3.40-3.51 (m,2H, NCH₂CH₂S), 4.07-4.19 (m, 1H, α H cys), 5.74 (d, J=7.6 Hz, 1H, NHcys), 6.35-6.45 (m, 1H, NHCH₂), 7.17-7.33, 7.38-7.47, 7.53-7.62,7.89-7.96 (4m, 20H, aromatic H). MS: (FAB⁺/G-T) m/z 597 (M+H)⁺.

Analysis: C₃₅H₃₆N₂O₃S₂ (596) Calc. %: C 70.47 H 6.04 N 4.70 Found %:70.14 6.10 4.792.40. N-(N-Isobutyryl-L-cysteinyl)-S-benzoylcysteamine (I-219)

This compound is obtained by the S-detritylation of 17 (2.68 mmol). Theprotocol used is the same as that described in example 1 for thesynthesis of I-152. After the various treatments, a gum is collected,which gum is purified by flash chromatography on a silica gel column(eluent: CH₂Cl₂/ether 25%). I-219 is isolated in the form of a gumwhich, after trituration in hexane, provides a colorless powder(Yd=58%). R_(f) (CH₂Cl₂/ether, 6/4): 0.35. M.p.=127-130° C. [α]_(D)²⁰=−20.8° (c 1.06, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.17, 1.18 (2d, J=2×6.9 Hz, 2×3H, C(CH₃)₂), 1.59(dd, J=7.5 and 10.3 Hz, 1H, SH), 2.44 (app. hept, J=6.9 Hz, 1H,CH(CH₃)₂), 2.70 (ddd, J=6.5, 10.3 and 13.8 Hz, 1H, β Ha cys), 3.07 (ddd,J=4.2, 7.5 and 13.8 Hz, 1H, Hb cys), 3.17-3.34 (m, 2H, NCH₂CH₂S),3.53-3.64 (m, 2H, NCH₂CH₂S), 4.62 (ddd, J=4.2, 6.5 and 8.0 Hz, 1H, α Hcys), 6.47 (d, J=8.0 Hz, 1H, NH cys), 6.80-6.91 (m, 1H, NHCH₂),7.42-7.53, 7.56-7.65, 7.92-8.01 (3m, 5H, aromatic H). MS: (FAB⁺/G-T) m/z709 (2M+H)⁺, 355 (M+H)⁺.

Analysis: C₁₆H₂₂N₂O₃S₂ (354) Calc. %: C 54.23 H 6.21 N 7.91 Found %:54.20 6.18 7.942.41. N-(N-Isobutyryl-S-acetyl-L-cysteinyl)-S-benzoylcysteamine (I-220)

The S-acylation of I-219 (0.25 mmol) with acetic anhydride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum is purified by flash chromatography on a silica gelcolumn (eluent: CH₂Cl₂/ether 15%). I-220 is isolated in the form of agum which, after trituration in hexane, provides a colorless powder(Yd=70%). R_(f) (CH₂Cl₂/ether, 7.5/2.5): 0.43. M.p.=174-176° C. [α]_(D)²⁰=−17.6° (c 0.91, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.13, 1.14 (2d, J=2×6.9 Hz, 2×3H, C(CH₃)₂),2.29-2.45 (m, 1H, CH(CH₃)₂), 2.32 (s, 3H, SCOCH₃ partially concealingthe m at 2.29-2.45), 3.18-3.38 (m, 4H, NCH₂CH₂S, CH₂ cys), 3.45-3.62 (m,2H, NCH₂CH₂S), 4.50-4.62 (m, 1H, α H cys), 6.39 (d, J=7.1 Hz, 1H, NHcys), 6.88-6.98 (m, 1H, NHCH₂), 7.42-7.52, 7.55-7.64, 7.92-8.01 (3m, 5H,aromatic H). MS: (FAB⁺/G-T) m/z 793 (2M+H) ⁺,397 (M+H)⁺.

Analysis: C₂₄H₃₂N₂O₄S₂ (396) Calc. %: C 54.54 H 6.06 N 7.07 Found %:54.46 6.02 7.082.42. N-(N,S-Bisisobutyryl-L-cysteinyl)-S-benzoylcysteamine (I-221)

The S-acylation of I-219 (0.25 mmol) with isobutyryl chloride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum, after triturations in hexane, provides a colorlesspowder which is homogeneous by TLC with a yield of 86%. R_(f)(CH₂Cl₂/ether, 7.5/2.5): 0.42. M.p.=141-143° C. [α]_(D) ²⁰=−9° (c 1.11,CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.13, 1.14 (2d, J=2×6.9 Hz, 2×3H, C(CH₃)₂ ofN-i-but), 1.18 (d, J=6.9 Hz, 6H, C(CH₃)₂ of S-i-but), 2.37 (app. hept,J=6.9 Hz, 1H, CH(CH₃)₂ of N-i-but), 2.75 (app. hept, J=6.9 Hz, 1H,CH(CH₃)₂ of S-i-but), 3.16-3.38 (m, 4H, CH₂ cys, NCH₂CH₂S), 3.42-3.64(m, 2H, NCH₂CH₂S), 4.47-4.58 (m, 1H, α H cys), 6.42 (d, J=7.0 Hz, 1H, NHcys), 6.87-7.01 (m, 1H, NHCH₂), 7.41-7.50, 7.55-7.63, 7.93-8.01 (3m, 5H,aromatic H). MS: (FAB⁺/G-T) m/z 849 (2M+H)⁺, 425 (M+H)⁺.

Analysis: C₂₀H₂₈N₂O₄S₂ (424) Calc. %: C 56.60 H 6.60 N 6.60 Found %:56.67 6.62 6.632.43. N-(N-Isobutyryl-S-pivaloyl-L-cysteinyl)-S-benzoylcysteamine(I-222)

The S-acylation of I-219 (0.25 mmol) with pivaloyl chloride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum is purified by flash chromatography on a silica gelcolumn (eluent: CH₂Cl₂/ether 20%). I-222 is isolated in the form of agum (Yd=50%) which, after trituration in hexane, provides a colorlesspowder. R_(f) (CH₂Cl₂/ether, 5/5): 0.55. M.p.=112-114° C. [α]_(D)²⁰=+4.6° (c 1.08, CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.12, 1.13 (2d, J=2×6.9 Hz, 2×3H, C(CH₃)₂), 1.22(s, 9H, C(CH₃)₃), 2.36 (app. hept, J=6.9 Hz, 1H, CH(CH₃)₂), 3.18-3.36(m, 4H, CH₂ cys, NCH₂CH₂S), 3.45-3.62 (m, 2H, NCH₂CH₂S), 4.48-4.60 (m,1H, α H cys), 6.48 (d, J=7.2 Hz, 1H, NH cys), 6.96-7.08 (m, 1H, NHCH₂),7.40-7.51, 7.54-7.63, 7.92-8.01 (3m, 5H, aromatic H). MS: (FAB⁺/G-T) m/z877 (2M+H)⁺, 439 (M+H)⁺.

Analysis: C₂₁H₃₀N₂O₄S₂ (438) Calc. %: C 57.53 H 6.85 N 6.39 Found %:57.31 6.86 6.342.44. N-(N-Isobutyryl-S-benzoyl-L-cysteinyl)-S-benzoylcysteamine (I-223)

The S-acylation of I-219 (0.26 mmol) with benzoyl chloride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum is purified by flash chromatography on a silica gelcolumn (eluent: CH₂Cl₂/ether 20%). I-223 is isolated in the form of agum (Yd=84%) which, after trituration in hexane, provides a colorlesspowder. R_(f) (CH₂Cl₂/ether, 7:3): 0.41. M.p.=154-156° C. [α]_(D)²⁰=+7.6° (c 0.92; CHCl₃).

¹H NMR (CDCl₃) δ ppm: 1.10, 1.11 (2d, J=2×6.9 Hz, 2×3H, C(CH₃)₂), 2.38(app. hept, J=6.9 Hz, 1H, CH(CH₃)₂), 3.19-3.28 (m, 2H, NCH₂CH₂S),3.43-3.64 (m, 4H, CH₂ cys, NCH₂CH₂S), 4.58-4.71 (m, 1H, α H cys), 6.55(d, J=7.2 Hz, 1H, NH cys), 6.92-7.01 (m, 1H, NHCH₂), 7.39-7.50,7.53-7.64, 7.91-7.99 (3m, 10H, aromatic H). MS: (FAB⁺/G-T) m/z 917(2M+H)⁺, 459 (M+H)⁺.

Analysis: C₂₃H₂₆N₂O₄S₂ (458) Calc. %: C 60.26 H 5.68 N 6.11 Found %:60.22 5.67 6.002.45. N-(N-Acetyl-S-trityl-L-cysteinyl)thiazolidine (18)

The coupling reaction of 7 (2 mmol) with thiazolidine is carried outaccording to method A described in the first synthetic route (example1). After returning to ambient temperature, stirring is continued for 12h. The reaction medium is then diluted with 50 ml of AcOEt, then washed(water, 70 ml; ice-cold saturated sodium bicarbonate, 50 ml; water, 2×50ml), dried over sodium sulfate and evaporated to dryness under vacuum.The colorless foam obtained is subsequently purified by flashchromatography on a silica gel column (eluent: AcOEt/petroleum ether30%). 18 is isolated in the form of a colorless gum with a yield of 80%.R_(f) (AcOEt/petroleum ether, 8/2): 0.40. Crystallizes from MeOH incolorless needles. M.p.=197-198° C. [α]_(D) ²⁰=+0.86° (c 1.16, CHCl₃).

¹H NMR (CDCl₃) δ ppm (isomeric mixture, 5.2/4.8: 1.95 (s, 3H, NCOCH₃),2.48-2.66 (m, 2H, CH₂ cys), 2.88-3.02 (m, 2H, H5, H5′ Thz), 3.24-3.34,3.64-3.75, 3.77-3.86 (3m, 2H, H4, H4′ Thz), 3.96, 4.41 and 4.45, 4.54(2×2d, J=2×8.8 and 2×10.3 Hz, 2H, H2, H2′ Thz), 4.60-4.69 (m, 1H, α Hcys), 6.05-6.15 (m, 1H, NH cys), 7.18-7.34, 7.36-7.44 (2m, 15H, aromaticH). MS: (FAB⁺/G-T) m/z 953 (2M+H)⁺, 477 (M+H)⁺.

Analysis: C₂₇H₂₈N₂O₂S₂ (476) Calc. %: C 68.07 H 5.88 N 5.88 Found %:67.97 5.84 5.892.46. N-(N-Acetyl-L-cysteinyl)thiazolidine (I-212)

This compound is obtained by the S-detritylation of 18 (0.77 mmol). Theprotocol used is the same as that described in example 1 for thesynthesis of I-152. After stirring overnight at ambient temperature, asolution is obtained. This solution is evaporated to dryness undervacuum and the pasty residue obtained is coevaporated with toluene (3×5ml) and then washed with 4×15 ml of ether to provide the correspondingsilver sulfide in the form of a yellow powder. This sulfide issubsequently treated in the same way as that described in example 1.After the various treatments, a translucent gum is collected, which gumis purified by flash chromatography on a silica gel column (eluent:AcOEt). I-212 is isolated in the form of a colorless gum with a yield of64%. R_(f) (AcOEt/MeOH, 9.7/0.3): 0.40. Crystallizes from anAcOEt/hexane mixture in the form of colorless needles. M.p.=90-91° C.[α]_(D) ²⁰=−31° (c 1, CHCl₃).

¹H NMR (CDCl₃) δ ppm (isomeric mixture, 5.8/4.2): 1.55 (app. t, J=8.9Hz, 1H, SH), 2.02 (s, 3H, NCOCH₃), 2.75-2.84, 2.85-2.95 (2m, 2H, CH₂cys), 2.99-3.18 (m, 2H, H5, H5′ Thz), 3.79-4.03 (m, 2H, H4, H4′ Thz),4.57, 4.63 and 4.71 (2d, J=2×10.4 and 1 app. s, 2H, H2, H2′ Thz),4.94-5.04 (m, 1H, α H cys), 6.41-6.52 (m, 1H, NH cys). MS: (FAB⁺/G-T)m/z 235 (M+H)⁺.

Analysis: C₈H₁₄N₂O₂S₂ (234) Calc. %: C 41.03 H 5.98 N 11.97 Found %:41.12 6.01 11.962.47. N-(N,S-Bisacetyl-L-cysteinyl)thiazolidine (I-213)

The S-acylation of I-212 (0.26 mmol) with acetic anhydride is carriedout according to the general method described in example 2. The reactionmixture is subsequently treated according to the protocol described forthe synthesis of I-177. After the various treatments, a gum iscollected, which gum is purified by flash chromatography on a silica gelcolumn (eluent: AcOEt/petroleum ether 10%). I-213 is isolated in theform of a gum (Yd=80%) which crystallizes from an AcOEt/petroleum ethermixture in colorless needles. R_(f) (AcOEt): 0.3, M.p.=107-108° C.[α]_(D) ²⁰=+6.6° (c 1.36, CHCl₃).

¹H NMR (CDCl₃) δ ppm (isomeric mixture, 5.9/4.1): 2.01 (s, 3H, NCOCH₃),2.36 (s, 3H, SCOCH₃), 2.97-3.20, 3.26-3.29, 3.30-3.33 (3m, 4H, CH₂ cys,H5, H5′ Thz), 3.73-3.81, 3.82-3.89, 3.96-4.06 (3m, 2H, H4, H4′ Thz),4.49, 4.61, 4.71, 4.81 (2×2d, J=2×10.3 and 2×8.9 Hz, 2H, H2, H2′, Thz),4.93-5.04 (m, 1H, α H cys), 6.39-6.50 (m, 1H, NH cys). MS: (FAB⁺/G-T)m/z 553 (2M+H)⁺, 277 (M+H)⁺.

Analysis: C₁₀H₁₆N₂O₃S₂ (276) Calc. %: C 43.48 H 5.80 N 10.14

EXAMPLE 3 Demonstration of Antiviral Activity of Compounds Obtained inExamples 1 and 2

3.1. Preamble

The manipulations of infectious material have been performed in a L3type high security laboratory.

In order to approximate as closely as possible to physiopathologicalconditions, all the studies were carried out using MDM, PBMC or PBLprimary cultures obtained from healthy blood donors.

In all the experiments, the effects of the novel molecules were comparedwith those of the reference molecules: NAC or MEA.

3.2. Isolation, Culturing and Activation of the Cells

3.2.1. Culture Media

The medium A is composed of RPMI 1640 cell culture medium (LifeTechnologies) supplemented with 10% of fetal calf serum (FCS, BoehringerMannheim) heat decomplemented at 56° C. for 30 min, of 2 mM ofL-glutamine (Boehringer Mannheim) and of a 100 μg/ml solution of 3antibiotics (penicillin, streptomycin and neomycin; PSN, LifeTechnologies). The medium B is composed of medium A supplemented with 20IU/ml of recombinant human IL-2 (Boehringer Mannheim).

3.2.2 Isolation of the Peripheral Blood Mononuclear Cells

The PBMCs are separated from the other components which appear in theblood by centrifugation on a Ficoll gradient (MSL 2000, Eurobio): 30 mlof blood from a healthy donor, diluted to a third, are deposited on a 20ml cushion of Ficoll. After centrifuging for 20 min at 850 g, the bandof PBMC is removed and then washed twice in RPMI 1640, aftercentrifuging for 10 min at 750 g and 5 min at 400 g.

3.2.3. Isolation of the Monocytes and Lymphocytes

The monocytes and the lymphocytes are isolated from the PBMCs bycountercurrent elutriation according to the protocol described by C.Figdor et al. (Cell. Biophys., 1983, 5, 105-118). The two cellpopulations thus separated are immunophenotyped and then analyzed usinga flow cytometer (FACScan, Becton Dickinson). The purity of themonocytes and PLBs thus obtained is greater than or equal to 95%.

3.2.4. Culturing and Activation of the Cells

A million monocytes in 1 ml of culture medium A are distributed in eachwell of a 48 well plate (Becton-Dickinson). The monocytes are left todifferentiate into macrophages for 7 days. The macrophages thusdifferentiated are maintained in culture in medium A.

For some experiments, the PBMCs and the PBLs are activated for 48 h with1 μg/ml of a mitogen, PHA-P (Difco Laboratories). The PBMCs and the PBLsare cultured in medium A (quiescent) or B (activated). The cells arecultured at 37° C. in an atmosphere saturated with moisture under 5% ofCO₂. The culture supernatants are removed and the culture media arereplaced every three or four days. At each renewal of the culture media,the cell viability is evaluated by coloring with trypan blue or bymicroscopic observation.

3.3. Evaluation of the Antiviral Activity of I-152 and of itsDerivatives

3.3.1. Preparation of the Compounds

During the first series for evaluation of the antiviral activity andduring the study of the mechanism of action of I-152, I-152 and thereference products were dissolved in medium A. The molecules wereresuspended at stock concentrations (NAC: 20 mM, MEA and I-152: 10 mM)and were stored at −80° C. The dilutions were subsequently preparedextemporaneously in medium A.

During the second series for evaluation of the antiviral activity, I-152and its derivatives (insoluble in medium A) were dissolved in DMSO andthen diluted in medium A. The concentration of DMSO during this study is1.5%. The solutions and the dilutions were prepared extemporaneously inorder to avoid or reduce oxidation of I-152 by DMSO to its disulfide.

3.3.2. Virus and Infection of the Cells

The MDMs were infected with the reference isolate with macrophagetropism, HIV-1/Ba-L. The PBMCs and the PBLs were infected with thereference isolate with lymphocyte tropism HIV-1 LAI. The viral stockwere formed by amplifying these strains in vitro using umbilical bloodmononucleated cells (UBM(s) preactivated with 1 μg/ml of PHA-P andcultured in medium A supplemented by 20 IU/ml of IL-2. In order toremove the soluble factors, such as cytokines, the culture supernatantswere ultracentrifuged at 360,000 g for 5 min, and the pellets wereresuspended in RPMI 1640. The viral stocks thus formed were subsequentlytitred using PBMCs activated with PHA-P. The TCID50s (50% Tissue CultureInfectious Dose) were calculated using the Karber formula.

A million MDMs were infected with 10,000 TCID50s of the HIV-1/Ba-Lstrain. This amount of virus corresponds to a multiplicity of infection(m.o.i.) equal to 0.01. The excess of virus is removed 24 h after bywashing with cells using RPMI 1640. The PBLs and the PBMCs were infectedwith 10,000 TCID50s of the HIV-1 LAI strain (moi=0.01). The cells werewashed at the end of the second day of infection.

3.3.3. Assaying the Viral Replication in the Culture Supernatants

3.3.3.1 Assaying the Reverse Transcriptase (RT) Activity

The viral replication is measured by assaying the RT activity in theculture supernatants according to the technique described by F. Rey etal. (Biochem. Biophys. Res. Comm., 1984, 121, 126-133). Theradioactivity incorporated during the extension of the complementarystrand of a poly-rA synthetic matrix in the presence of an oligo-dT₁₂₋₁₈primer and of a radiolabeled substrate,[³H-methyl]thymidine-5′-triphosphate ([³H]TPP), makes it possible toassay the enzymatic activity of RT. 400 μM of supernatant areultracentrifuged at 360,000 g for 5 min. The RT is released by lysis ofthe viral pellet in 20 μl of NTE-Triton (100 mM NaCl, 10 mM Tris, 1 mMEDTA, 0.1% Triton X-100). These 20 μl are subsequently incubated with 40μl of the following reaction mixture: 62.5 mM Tris, pH 7.8; 25 mM KCl;6.25 mM MgCl₂; 1.25 mM dithiothreitol (DTT); 2.5×10⁻³ ODU oligo-dT₁₂₋₁₈and poly-rA, 5.55×10⁻³ TBq {³H]TTP. After one hour at 37° C., theenzymatic reaction is halted and the newly synthesized strands areprecipitated for 20 min at 4° C. by the addition of 1 ml of sodiumpyrophosphate (NaPP), of 50 μl of yeast DNA (0.1 mg/ml in 5%trichluoroacetic acid (TCA)), and of 4 ml of 20% TCA. The mixture isfiltered using a cellulose acetate membrane (Millipore) which retainsthe radiolabeled poly-dT chains. The filter is washed using 20 ml of 5%TCA and the residual water is removed by addition of 25 μl of 70%ethanol. The filter is dried in an oven for 10 minutes at 80° C. and isthen introduced into vials containing 8 ml of liquid scintillant. The βradioactivity is quantified by means of a scintillation counter (PackardBell). The results are expressed in pM of [³H]-TMP incorporated/h/ml ofsupernatant or, more simply, in cpm/h/ml.

3.3.3.2 Assaying the P25 Protein

The assaying of the p25 protein is carried out using the ELISA kit fromDuPont de Nemours. 200 μl of the culture supernatant to be tested areplaced in a well of a microtitration plate. The addition of 20 μl oflysis buffer releases the viral proteins in the medium. The releasedantigen attaches to a mouse anti-p25 monoclonal antibody immobilized atthe bottom of the wells. After incubating for 2 h at 37° C., 3 washingoperations using 5 ml of washing buffer are carried out and then 100 μlof a biotinylated polyclonal antibody which reacts with the immobilizedantigen are incubated for 1 h at 37° C. The series of 3 washingoperations in the same buffer and with the same volume is again carriedout before the addition over 15 min at 37° C. of 100 μl ofRaifort-streptavidin peroxidase conjugate which will make it possible toamplify the colorimetric reaction. The complex formed is revealed, after3 washing operations using 5 ml of the washing buffer of the kit, with100 μl of o-phenylenediamine (OPD) dihydrochloride. After incubating for30 min at ambient temperature, the reaction is halted by the addition of100 μl of 4N sulfuric acid. The OD of the coloring thus obtained is readat 490 mm. This absorbence is directly proportional to the amount ofattached antigen. The linear relationship relating the O.D. to the p25concentration is established by virtue of a standard range produced froma recombinant p25 solution.

3.3.4. Analysis of the Results and Determination of the 50% EffectiveDoses

The 50% effective doses (ED50) are calculated from the cumulative RTactivities using the software “Dose-effects analysis withmicrocomputers” developed by J. Chou & T. C. Chou.

3.3.5. Measurement of the Cell Viability

These tests are carried out systematically in parallel with theevaluation of the antiviral activity. In view of the oxidation/reductioncapacity of the molecules tested, the test with the tetrazolium salt,which measures the activity of mitochondrial dehydrogenases, cannot beused.

3.3.5.1. Measurement Using an Exclusion Dye, Trypan Blue

The nonadherent cells, such as the PBMCs and the PBLs, are counted usinga Malassez cell and an exclusion dye, trypan blue (TB). 25 μl of thecell suspension are added to 475 μl of TB. This count is carried outafter the isolation of the PBMCs and the PBLs, before the plating, andat each replacement of the culture medium.

3.3.5.2. Measurement Using a Vital Dye, Neutral Red

Neutral red (NR) is a vital dye which makes it possible to measure theviability of adherent cells, such as MDMs. 600 μl of culture supernatantare removed and replaced with 400 μl of an NR solution (0.001% m/v inphosphate buffer, PBS, Boehringer Mannheim), filtered at 0.45 μm. Thecells were incubated for 1 h at 37° C. and are subsequently washed (2×1ml of PBS). The cells are then lysed at −20° C. with 200 μl of a 50%ethanol mixture comprising 1% of glacial acetic acid. The OD is measuredtwice with 100 μl of solution using a spectrophotometer.

3.4. Study of the Mechanism of Action of I-152

3.4.1. Quantification of the Proviral DNAs by PCR

I-152 is composed of MEA and NAC, which are capable of interacting withthe early phases of the biological cycle of HIV. It is therefore capableof decreasing the integration of the provirus into the cell genome. Inorder to measure these effects, the proviral DNAs were quantified byPCR. The cells were lysed using 1 ml of the following lysis solution: 10mM Tris HCl pH 8; 100 mM of EDTA pH 8; 0.5% of sodium dodecyl sulfate(SDS); 20 μg/ml of DNAse-free bovine pancreatic RNAse. 200 μg/ml ofproteinase K are subsequently added to this suspension. The DNAs weresubsequently extracted using 1 ml of an ice-cold saturated phenolsolution and 1 ml of a phenol/Chisam solution.

The viral DNAs were then amplified by means of specific primers of thegag gene (SK01/SK39) and of a standard range of the 8E5 line, achronically infected line, the cells of which carry a proviral copy. Theβ-globin gene was used as reporter gene in order to make sure of thequality of the DNA extraction.

3.4.2. Acellular Test of the Enzymatic Activity of RT

The I-152 molecule is composed of NAC and MEA, which are capable ofinhibiting the activity of RT (A. Bergamini et al., J. Clin. Invest.,1994, 93, 2251-2257). For this reason, the inhibiting capacity of I-152with respect to the RT activity was measured using an acellular test.This test was carried out according to the protocol described above (§3.3.3.1.). In the reaction mixture, only the 20 μl of water are replacedby 20 μl of a concentration of I-152 or of the reference compounds.Heparin, known for inhibiting the activity of RT and of the other DNApolymerases, is used as positive control for inhibition.

3.4.3. Assaying the Total Glutathione

The method for assaying the total glutathione (GSH+GSSG) which we usedis an adaptation, to the MDM culture system, of that described by O. W.Griffith et al. (Anal Biochem., 1980, 106, 207-212). The assayings werecarried out 24 h after the beginning of the treatment by the variouscompounds. A million cells are washed three times in PBS and then lysedwith 150 μl of a lysis buffer (0.1M phosphate pH=7.4; 0.15M NaCl; 0.1%BSA, 0.01% Azide, 0.1% Triton X-100; 0.05% 5′-sulfosalicylic acid). Thestandard range in doubling dilutions ranges from 50 μl to 1.5 nM of GSSGor of GSH. The test is carried out in triplicate. 85 μl of 0.6 mM NADPH,25 μl of 6 mM DTNB and 130 μl of pure water are added to the samples.The latter are incubated at 30° C. for 10 min. At the time of reading,20 μl of 1 U/ml GSSG reductase are added to all the wells. Theabsorbence is measured with the long pathway at 412 nm. Theconcentration of total glutathione is subsequently determined withrespect to the values of the calibration curve, produced in parallelwith the assaying, and by extrapolation in the region of the linear partof the curve.

EXAMPLE 4 Anti-HIV Activity of I-152 With Respect to Macrophages

4.1 Antiviral Activity of I-152 With Respect to MDMs InfectedExtemporaneously

4.1.1. 50, 70 and 90% Effective Doses and Cytotoxicity of I-152

The cells of the macrophage line play a major role in oxidative“processes”. The pro-GSH molecule, I-152, was therefore tested withrespect to MDMs infected with the VIH-1/Ba-L strain. I-152, afterintracellular metabolization, is capable of releasing NAC, MEA andcysteine (FIG. 1).

We compared the activities of I-152 with those of its two components,NAC and MEA, in our experimental system. I-152 has a strong antiviralactivity superior to that of NAC or of MEA (FIG. 2, table 1). Theinhibiting concentrations of NAC, of MEA and of I-152 are respectivelyequal to 9.4 mM, 300 μM and 50 μM. In the light of these figures, I-152therefore appears to be 6 and 188 times more effective than MEA and NAC.However, these values do not reflect the difference between these threeproducts. This is because, at antiviral doses, NAC and MEA are cytotoxicwhereas I-152 is not cytotoxic. Thus, NAC is cytotoxic from 10 or 15 mM(FIG. 3: NAC 15 mM: 70% cytotoxicity; NAC 40 mM: 91%), and MEA decreasesthe viability of MDMs by 65% at the concentration of 500 μl (FIG. 3). Onthe other hand, I-152, even at doses 10 times greater than its 50%effective dose (ED), is not cytotoxic (FIG. 3: 500 μM).

4.1.1.2. Effect of I-152 on the Production of the Major Protein of theViral Nucleocapsid

The viral replication can be measured in the culture supernatants byassaying either the enzymatic activity of RT or the major protein of theviral nucleocapsid, p25. In infected MDM cultures, the inhibition of theproduction of the p25 protein is concomitant with that of the RTactivity (FIG. 4). These results therefore confirm the antiviraleffectiveness of I-152.

4.1.1.3. Effect of the Multiplicity of Infection on the AntiviralActivity of I-152

During our first experiments, the cells were infected using 10,000TCID50 (m.o.i.: 0.01). In order to measure the effects of the viral loadon the anti-HIV activity of I-152, the cells are infected, in a secondstep, using 1,000 TCID50 (m.o.i.: 0.001).

The antiviral activity of I-152 increases when the m.o.i. is decreased(FIG. 5) and the ED turns out to be decreased thereby (table II, 3 μlvs. 50 μM).

4.1.2. Antiviral Activity of I-152 With Respect to Preinfected MDMs

The treatment of the cells 7 days after infection confirms the antiviralactivity of I-152 (FIG. 6). This is because, at a dose of 500 μM, viralreplication is extinguished. However, under these experimentalconditions, the dose of 250 μl is ineffective. This shift in theinhibiting potential, observed between the various methods of treatment,suggests 1) that I-152 inhibits viral replication by probably combiningtwo mechanisms: an early and a late mechanism, and 2) that, at high dose(≧500 μM), inhibition of the late phase of the biological cycle issufficient.

4.2. Antiviral Activity of I-152 in Primary Cultures of Lymphocytes andof Peripheral Blood Mononuclear Cells

After having shown the antiviral activity of I-152 with respect to cellsof the macrophage line, its effects were measured with respect toperipheral blood lymphocytes (PBLs) and mixed population comprisingmonocytes/macrophages and lymphocytes, PBMCs. Furthermore, in order tomeasure the effects of cell activation on the antiviral activity ofI-152, the cells were or were not activated by a mitogen,phytohemagglutinin-P (PHA-P).

4.2.1. Viability of the PBMCs and PBLs Treated With I-152

The cell viability was measured in parallel with the assessment of theantiviral activity. At each replacement of the culture medium, the,viable cells were counted by means of an exclusion dye, trypan blue. Inthe PBMC and PBL cultures, I-152 is not cytotoxic. This is because theviability of the lymphocytes is not decreased in the PBL and PBMCcultures and, furthermore, in the latter, when the cells are not exposedto PHA-P, the monocytes differentiate into macrophages with apseudofibroblastic appearance. On the other hand, NAC and MEA arecytotoxic for both cell types from doses of 10 or 15 mM and 500 μMrespectively.

4.2.2. Antiviral Activity of I-152 With Respect to Quiescent PBMCs orPBMCs Activated by PHA-P, and Infected

The PBMCs, quiescent or activated by PHA-P, were infected by thereference strain with lymphocyte tropism HIV-1 LAI. All the culturingswere carried out in parallel and the drugs were maintained throughoutthe culturing.

In both cell populations, MEA and NAC do not inhibit viral replicationat known cytotoxic doses (results not presented). In the quiescentPBMCs, I-1.52 inhibits viral replication by 97 and 88% at doses of 250and 125 μl respectively and, in the activated PBMCs viral replication isdecreased by 42 and 25% at the same doses (FIG. 7).

4.2.3. Antiviral Activity of I-152 With Respect to Quiescent PBLs orPBLs Activated by PHA-P, and Infected With the HIV-1 LAI Strain

As in the PBMCs cultures, MEA and NAC do not have or have a very slightantiviral activity in PBLs (results not presented). On the other hand,I-152 inhibits viral replication in quiescent PBLs or PBLs activated byPHA-P (FIG. 8).

4.3. Anti-HIV Activity of I-152 With Respect to Tissue Macrophages

4.3.1. Isolation of Spleen Monocytes/Macrophages

The spleen is dissected and sieved and then the mononuclear cells areisolated using a density gradient, on a Ficoll cushion. Themonocytes/macrophages are obtained by adherence to the plastic. Themonocytes were allowed to differentiate into macrophages for 7 days, atwhich time they were infected.

4.3.2. Results

The monocyte/macrophage plays a deleterious role in the physiopathologyof infections by the HIV as it constitutes an important site forretroviral replication within tissues. As these tissues are not veryaccessible to current anti-HIV therapy, this cell population is regardedas a “reservoir”.

Retroviral replication within this cell population, and thus theeffectiveness of a molecule such as I-152, is conditioned partly by thelevel of cell differentiation. It was therefore important to make sureof the antiviral effectiveness of I-152 in monocytes/macrophages with adegree of maturation which can be different from that of MDMs. We havethus evaluated the antiviral activity of I-152 and its ability toregenerate the intracellular level of GSH in spleen macrophages. In thiscell populations, the HIV-1/Ba-L strain also replicates at high noise.The I-152 molecule has proved to be as effective in human spleenmacrophages as in macrophages derived from blood monocytes. This isbecause the concentration of 38 μM decreases HIV replication by 50% inthis spleen cell population (table III). Likewise, as in the macrophagesderived from blood monocytes, I-152 increases the intracellular level ofGSH in a dose-dependent way which can be saturated from 250 μl (FIG.15). It should be noted that, in the spleen macrophages, as in themacrophages derived from blood monocytes, infection by HIV leads to adeficiency of GSH (MDM; figure16).

4.4. Mechanism of Action of I-152

4.4.1. Effect of I-152 on the Integration of the Proviral Genome Withinthe Cell Genome

Cell culture experiments would suggest that I-152 inhibits HIVreplication by probably combining two mechanisms, the one early and theother late. In order to measure the effects of this molecule on theearly phases of the biological cycle of the HIV, the integration of theprovirus within the cell genome was quantified by PCR. I-152 inhibitsthe integration of the provirus. This inhibition is much greater thanthat induced by NAC or MEA. On the other hand, it is not perfect, unlikethat induced by 10 μl AZT (FIG. 9).

4.4.2. Effect of I-152 on the Enzymatic Activity of RT in an AcellularSystem

In view of the preceding results and because MEA is capable ofinhibiting the activity of RT, the effects of I-152 on the activity ofthis enzyme were measured in an acellular system. The experiment wascarried out in duplicate and heparin was used as control of theinhibition of the activity RT. In both cases, heparin at theconcentration of 1 mg/ml completely inhibits the RT activity of theHIV-1 LAI isolate. This inhibition is dose-dependent. Likewise, MEAdecreases by 29±1% (exp. 1, FIG. 10) and 48±4% (exp. 2 not presented) ata dose of 500 μM. On the other hand, NAC and I-152 do not exhibit aninhibiting activity with respect to the RT of the HIV in ourexperimental system (FIG. 10). These results confirm that I-152, byreleasing MEA, can interact with an early stage of the biological cycleof the HIV.

4.4.3. Effect of I-152 on the Intracellular Concentration of Glutathione

The late effects of I-152 are probably the more direct consequence ofits proglutathione activity. In order to make sure of this activity inlymphocytes and macrophages, the intracellular concentrations of totalglutathione (GSH+GSSG) were determined in PBMC cultures (FIG. 11). Ofthe two reference molecules, NAC is better at increasing theintracellular level of glutathione in quiescent PBMCs. This is becauseMEA does not significantly alter the intracellular level of glutathione,at the very least at the doses tested. In our experiments, the doses ofNAC used are higher than those of MEA, which may explain the differenceobserved in the intracellular level of glutathione.

I-152 increases the intracellular level of glutathione. This increasefollows a bell-shaped curve with an optimum at 25 and 125 μM. The doseof I-152 of 125 μM increases the cellular concentration up to 2.64±0.13μM. This intracellular concentration is equivalent to that obtained withthe dose of NAC of 15 mM. On considering the concentrations of moleculessupplementing the culture medium, I-152 is 120 times more active thanNAC, It should be noted that the intracellular concentrations ofglutathione decrease at high concentrations, at the very least for MEA(500 μM) and I-152 (250 μM). This phenomenon is probably the consequenceof the processes for the regulation of glutathione.

4.5 Antiviral Activity of the S-Acylated Derivatives of I-152: I-176,I-177 and I-178, With Respect to MDMs Infected Extemporaneously

During a second series of experiments, the antiretroviral activities ofthe I-152 derivatives were tested with respect to MDMs infected by theHIV-1/Ba-L strain (FIG. 12). These molecules are lipophilic and werethus dissolved in dimethyl sulfoxide (DMSO) and then diluted. I-152,which acts as reference, was treated in the same way. Under theseexperimental conditions, I-152 proved to be the most effective ininhibiting viral replication. However, it should be noted that DMSOdecreases its antiviral effectiveness and that the standard deviation ishigh as the antiviral activity was decreased with respect to two of thethree wells of the culture triplicate. The compound I-176 is slightlytoxic at 150 μM, which may also explain the variability within theculture triplicate and the size of the standard deviation observed. Todate, none of the S-acylated derivatives has shown, in vitro, anactivity superior to that of the mother molecule. Nevertheless, I-177and I-178 have activities similar to that of I-152, and, in view of theimportance of the latter, these two molecules, which are morelipophilic, are to be retained. It may have advantages in the case ofclinical experimentations. This is because they can have pharmaceuticaldosage forms and methods of administration different from those ofI-152.

EXAMPLE 5 Effects of I-152 on the Intracellular Concentration of GSH

5.1. Materials and Methods

With the exception of materials and methods presented below, theprocedures are similar to those described in the preceding examples.

The intracellular GSH was assayed by means of the assay kit sold byCayman. This method for assaying glutathione is an adaptation of thatdescribed by O. W. Griffith et al. (1980).

The compound I-152 and its derivatives are prepared extemporaneously bybeing dissolved in dimethyl sulfoxide (DMSO) at the concentration of 100mM. They are subsequently stored at −20° C. after having been diluted 10fold in phosphate buffer (PBS).

5.2. Results

The effects of the compound I-152 in increasing the intracellularconcentration of GSH were demonstrated in cells not exposed to anoxidative stress. The present results show that this molecule is alsocapable of regenerating an abnormally low intracellular level. This isbecause the infection of macrophages derived from human blood monocytes(MDMs) leads to an intracellular deficiency of GSH (FIG. 13); thisdeficiency is accompanied by an increase in the expression of theenzymes involved in maintaining this level and by an increase in8-isoprostane, evidence of an oxidative stress (results not presented).

EXAMPLE 6 Effect of I-152 on the Macrophage Synthesis of TNF-α

6.1. Materials and Methods

With the exception of materials and methods presented below, theprocedures are similar to those described in the preceding examples.

The synthesis of tumor necrosis factor (TNF-α) was modified in the MDMculture supernatants using the calorimetric kit sold by Cayman.

The compound I-152 and its derivatives are prepared extemporaneously bybeing dissolved in dimethyl sulfoxide (DMSO) at the concentration of 100mM. They are subsequently stored at −20° C. after having been diluted 10fold in phosphate buffer (PBS).

6.2. Results

Tumor necrosis factor (TNF-α), like the oxygen-comprising ornitrogen-comprising radical components, plays a major role in theapoptotic processes associated with infection the HIV. Furthermore, theradical components can promote the synthesis of this proinflammatorycytokine. For this reason, the effects of I-152 in decreasing thesynthesis of TNF-α were thus looked for in MDMs stimulated by abacterial lipopolysaccharide and interferon (IFN-γ). The compound I-152inhibits the synthesis of TNF-α and increases the concentration of GSHunder these experimental conditions (FIG. 14).

EXAMPLE 7 Potentiation of the Antiretroviral Activity of AZT by thecompound I-152

7.1. Materials and Methods

With the exception of materials and methods presented below, theprocedures are similar to those described in the preceding examples.

Viral replication was measured by assaying the reverse transcriptaseactivity in culture supernatants using the RetroSys kit from Innovagen,following the recommendations of the company.

The compound I-152 and its derivatives are prepared extemporaneously bybeing dissolved in dimethyl sulfoxide (DMSO) at the concentration of 100mM. They are subsequently stored at −20° C. after having been diluted 10fold in phosphate buffer (PBS).

7.2. Results

The pro-GSH compounds of the “I-152 family” are preferably administeredas adjuvants therapeutic for current antiretrovirals. The inventors havethus attempted to measure in vitro the effects of I-152 with respect tothe anti-HIV effectiveness of these molecules. This study was carriedout using macrophages derived from human blood monocytes infected by theHIV-1/Ba-L strain and according to the methodology described by J. and TC. Chou for quantifying the synergistic effects, additive orantagonistic, between two molecules.

In our experimental model, the compound I-152 potentiates the anti-HIVactivity of AZT. This is because the combination index (CI) is less than1, which testifies to a synergy between the two compounds tested (FIG.17).

EXAMPLE 8 Antiretroviral Activity of the Acylated Analogues of I-152 orof ITS Derivatives

8.1. Materials and Methods

With the exception of materials and methods presented below, theprocedures are similar to those described in the preceding examples.

The 50% effective doses (ED50) are calculated from the cumulative RTactivities using the software “Dose-effects analysis withmicrocomputers” developed by J. Chou and T. C. Chou.

The compound I-152 and its derivatives are prepared extemporaneously bybeing dissolved in dimethyl sulfoxide (DMSO) at a concentration of 100mM. They are subsequently stored at −20° C. after having been diluted 10fold in phosphate buffer (PBS).

8.2. Results

The anti-HIV activity of about twenty I-152 derivatives, in addition tothat of the S-acylated compounds I-176, I-177 and I-178, was evaluatedin the system of MDMs infected in vitro by the HIV-1/Ba-L strain. Thesecompounds, attempted, to some extent, to be more lipophilic; they arethus capable of making possible pharmaceutical dosage forms and methodsof administration different from those of I-152. Furthermore, thesederivatives constitute the first link in the structure-activity studywhich should make it possible to identify important residues in theactivity of this family of compounds. The “stock” solutions of thesecompounds were treated with ultrasound in order to improve thesolubility of the products.

Nine compounds demonstrated a significant antiviral activity of the sameorder as that of I-152 (tables IV and V). The values obtained with thecompound I-152 which are presented in these two tables perfectlyillustrate the reproducibility of the anti-HIV effects of I-152 from oneexperiment to another and/or and from one cell donor to another. This isbecause the values presented in table III result from a first experimentcarried out using cells from one donor and those presented in table IVfrom a second experiment carried out using cells from another donor.

In conclusion, the compound I-152 or one of its analogues or of itsderivatives constitutes an excellent adjuvant therapeutic for currentantiretrovirals, such as AZT, and optionally for other classes ofantiretroviral molecules currently used in human clinical treatment(i.e. nonnucleoside reverse transcriptase inhibitors and viral proteaseinhibitors) while being capable of reorganizing both damage to theimmune system and that to the oxidative metabolism. Furthermore, theseresults have demonstrated the biological activities of other moleculeswhich are derivatives and/or analogues of I-152, the subject matter ofthe present invention; which henceforth constitutes a family ofmolecules which are biologically active and which can potentially beused as therapy.

The ability to increase the intracellular level of GSH and also toregenerate this tripeptide under conditions of stress where referencemolecules, such as NAC and MEA, are ineffective is also highlyadvantageous. These powerful antioxidant and probably antiapoptotic (ifit is considered that the radical components and TNF-α play a major rolein these deleterious processes) activities are in favor of a promotionof the compound I-152 in other noninfectious situations.

REFERENCES

-   Abrams, 1991, Am. J. Med., 91, 106-112.-   Barnett et al., 1969, J. Amer. Chem. Soc., 91, 2358-2369.-   Bergamini et al., 1994, J. Clin. Invest., 93, 2251-2257.-   Bosegaard et al., 1993, J. Pharmacol. Exp. Ther., 265, 1239-1244.-   Brückner et al., 1989, J. Chromatogr., 476, 73-82.-   Figdor et al., 1983, Cell, Biophys., 5, 105-118.-   Griffith et al., 1980, Anal. Biochem., 106, 207-212.-   Heller et al., 1997, Advances in Pharmacology, 38, 629-638.-   Herzenberg et al., 1997, Proc. Natl. Acad. Sci., 94, 1967-1972.-   Horowitz, 1991, Am. J. Med., 91, 113-117.-   Rabinovitch et al., 1992, Diabetologie, 35, 409-413.-   Rey et al., 1984, Biochem. Biophys, Res. Comm., 121, 126-133.-   Volante, 1981, Tetrahedron Lett., 22, 3119-3122.-   Wieland and Bokelman, 1952, Ann. Chem., 576, 20-34.-   Zee-cheng et al., 1970, J. Med. Chem., 13, 414-418.

1. A compound of general formula:

in which: R and R′ independently represent a linear or branched C₁-C₇alkyl radical or an aryl group which is unsubstituted or substituted byone or more radicals chosen from halogens, linear or branched C₁-C₃alkyl radicals and —OH radicals; R″ is hydrogen or a CO—R¹ group inwhich R¹ is a linear or branched C₁-C₇ alkyl radical or an aryl groupwhich is unsubstituted or substituted by one or more radicals chosenfrom halogens, linear or branched C₁-C₃ alkyl radicals and —OH radicals;and the dimers formed by a disulfide bridge from one and/or other of thetwo sulfur atoms of the compounds of general formula I composed of theR″ radicals or of the R′ CO— radicals of the two molecules, and thecorresponding thiazolidine forms.
 2. The compound as claimed in claim 1,characterized in that R is a methyl group (—CH₃).
 3. The compound asclaimed in claim 2, characterized in that R′ is a methyl group (—CH₃).4. The compound as claimed in claim 3, characterized in that R″ ishydrogen (Compound N-(N-acetyl-L-cysteinyl)-S-acetylcysteamine).
 5. Thecompound as claimed in claim 3, characterized in that R″ is an acetylgroup (—COCH₃) (CompoundN-(N,S-bisacetyl-L-cysteinyl)-S-acetylcysteamine).
 6. The compound asclaimed in claim 3, characterized in that R″ is an isobutyryl group(—COCH(CH₃)₂) (CompoundN-(N-acetyl-S-isobutyryl-L-cysteinyl)-S-acetylcysteamine).
 7. Thecompound as claimed in claim 3, characterized in that R″ is a pivaloylgroup (—COC(CH₃)₃) (CompoundN-(N-acetyl-S-pivaloyl-L-cysteinyl)-S-acetylcysteamine).
 8. The compoundas claimed in claim 2, characterized in that R′ is selected from theisopropyl group (—CH(CH₃)₃), the tert-butyl group (—C(CH₃)₃) and thephenyl group (—C₆H₅).
 9. The compound as claimed in claim 8,characterized in that R″ is selected from hydrogen (—H), the acetylgroup (—COCH₃), the isobutyryl group (—COCH (CH₃)₂), the pivaloyl group(—COC(CH₃)₃) or the benzoyl group (—CO—C₆H₅).
 10. The compound asclaimed in claim 1, characterized in that R is an isopropyl group(—CH(CH₃)₂).
 11. The compound as claimed in claim 10, characterized inthat R′ is selected from the methyl group (CH₃), the isopropyl group(—CH(CH₃)₂), the tert-butyl group (—C(CH₃)₃) and the phenyl group(—C₆H₅).
 12. The compound as claimed in claim 11, characterized in thatR″ is selected from hydrogen (—H), the acetyl group (—COCH₃), theisobutyryl group (—COCH (CH₃)₂), the pivaloyl group (—COC(CH₃)₃) or thebenzoyl group (—CO—C₆H₅).
 13. The compound as claimed in claim 8,characterized in that R″ is the trityl group.
 14. The compound asclaimed in claim 11, characterized in that R″ is the trityl group. 15.The compound as claimed in claim 1 to 14, characterized in that it is inthe thiazolidine form.
 16. A pharmaceutical composition for thetreatment of AIDS characterized in that it comprises a therapeuticallyeffective amount of the compound as claimed in claims 1 to 14 and apharmaceutically acceptable vehicle.
 17. A product comprising at leastone compound as claimed in one of claims 1 to 14 and at least onereverse transcriptase inhibitor as combination product for a use inantiviral therapy which simultaneous, separate or spaced out over time.18. The product as claimed in claim 17, characterized in that saidreverse transcriptase inhibitor is selected from3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyinozine (ddI),2′,3′-dideoxycytidine (ddC), (−)-2′,3′-dideoxy-3′-thiacytidine (3TC),2′,3′-didehydro-2′,3′-dideoxy-thymidine (d4T) and(−)-2′-deoxy-5-fluoro-3′-thiacytidine (FTC),(+)-5(S)-4,5,6,7-tetrahydro-8-chloro-5-methyl-6-(3-methyl-2-butenyl)imidazo-[4,5,1jk]-[1,4]benzodiazepin-2(1H)-thione(TIBO), 1-[2-hydroxyethoxy)methyl]-6-(pheylthio)thymine (HEPT),[2′,5′-bis-O-(tert-butyldimethylsilyl)-β˜D-ribofuranosyl]-3′-spiro-5″-(4″-amino-1″,2″-oxathiole-2″,2″-dioxide)thymine(TSAO), 2-(2-acetyl-methylanilino)-2-(2,6-dichlorophenyl)acetamide(α-APA), bis-(heteroaryl)piperazine [also referred to as<<rescriptor>>:1-(5-methanesulphonamido-1H-indol-2-yl-carbonyl)-4-[3-(1-methylethyl-amino)pyrodinyl]piperazinemonoethane sulphonate] (BHAP) or phosphonoformic acid (PFA).